UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

Date: June 29, 2015

SUBJECT: EDSP Weight of Evidence Conclusions on the Tier 1 Screening Assays for the List 1 Chemicals

PC Code: See table, Attachment A Decision No.: NA Petition No.: NA
Risk Assess ment T y pe: NA
TXR No.: See table

, Attachment A
MRIDNo.:NA DP Barcode: NA Registration No.: NA Regulatory Action: NA
Case No.: NA
CAS No.: NA
40CFR: NA

FROM: Greg Akerman 

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Health Effects Divison

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Environmental

# Fat**~~ fec** **~~ ~n** **~** Office of Pesticide Program

THR O UG H: Je ss Rowland

## ~ S

## ~~~ Deputy Divi s ion Director
Health Effects Division
Office of Pe s ticide Programs

TO: Jolene Trujillo Chemical Review Manager
Pe s ticide Re-Evaluation Division Office of Pesticide Programs Ver .A pr. 2010

EPA has completed its Weight of Evidence (WoE) assessment eval u ating results of the
Endocrine Screening Program (EDSP) Tier l screening assays for the List l chemicals. The WoE documents for the 52 chemicals are li sted in Attac hm ent A a l ong with the chemical
and report identifiers

.

Page I of2Page 2 of 2 Attachment A. EDSP List 1 Chemicals

Chemical Name PC Code TXR Number 2,4-D 030001 0057151
Abamectin 122804 0057152 Acephate 103301 0057153
Acetone 044101 0057154
Atrazine 080803 0057155
Benfluralin 084301 0057156
Bifenthrin 128825 0057157 Captan 081301 0057158 Carbaryl 056801 0057159
Carbofuran 090601 0057160
Chlorothalonil 081901 0057161 Chlorpyrifos 059101 0057162 Cyfluthrin 128831 0057163 Cypermethrin 109702 0057164
DCPA 078701 0057165
Diazinon 057801 0057166
Dichlobenil 027401 0057167
Dimethoate 035001 0057168
EPTC 041401 0057169
Esfenvalerate 109303 0057170 Ethoprop 041101 0057171
Fenbutatin-Oxide 104601 0057172
Flutolanil 128975 0057173 Folpet 081601 0057174 Glyphosate 417300 0057175 Imidacloprid 129099 0057176 Iprodione 109801 0057177 Isophorone 847401 0057178
Linuron 035506 0057179
Malathion 057701 0057180 Metalaxyl 113501 0057181 Methomyl 090301 0057182
Metolachlor 108801 0057183
Metribuzin 101101 0057184
MGK-264 057001 0057185 Myclobutanil 128857 0057186
Norflurazon 105801 0057150 o-Phenylphenol 064103 0057146 Oxamyl 103801 0057142
PCNB 056502 0057138
Permethrin 109701 0057149
Phosmet 059201 0057145 Piperonyl Butoxide 067501 0057141
Pronamide 101701 0057137 Propargite 097601 0057148 Propiconazole 122101 0057144 Pyriproxyfen 129032 0057140
Simazine 080807 0057136
Tebuconazole 128997 0057143 Tetrachlorvinphos (TCVP) 083701 0057147
Triadimefon 109901 0057139
Trifluralin 036101 0057135

### EDSP: WEIGHT OF EVIDENCE ANALYSI S OF POTENTIAL
INTERACTION WITH THE ESTROG EN, ANDROGEN OR THYROID
PATHWAYS

CHEMICAL: O -PHENYLPHENOL (o-PP)

O FFICE OF 

### P ESTICIDE 

### P ROGRAMS



### O FFICE OF 

### S CIENCE 

### C OORDINATION AND 

### P OLICY



### U.S. E NVIRONMENTAL 

### P ROTECTION 

### A GENCYO-PHENYLPHENOL FINAL

ii TABLE OF CONTENTS

Abbreviations...................................................................................................iii
Executive Summary..........................................................................................1

I. Introduction.......................................................................................4

II. Sources of Scientific Data and Technical Information......................................5

A. EDSP Tier 1 Screening Assays............................................................5

B. Other Scientifically Rele vant Information (OSRI)......................................5

III. Weight of Evidence (WoE) Evaluation.......................................................5

A. EDSP Tier 1 Screening Assays.............................................................8

B. Effects on Hypothalamic-Pituitary-Gonadal (HPG) Axis..............................9
1. Effects on Estrogen Pathway.....................................................9
2. Effects on Androgen Pathway..................................................13

C. Effects on Hypothalamic-Pituitary-Thyroidal (HPT) Axis...........................17

IV. Committee’s Assessment of Weight of Evidence..........................................20

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI ...................20

1. Tier 1 in vivo Assays.............................................................21

2. OSRI...............................................................................22

B. Estrogen Pathway...........................................................................24

C. Androgen Pathway.........................................................................24

D. Thyroid Pathway...........................................................................25

E. Conclusions.................................................................................25

V. EDSP Tier 2 Testing Recommendations....................................................25

VI. References.......................................................................................26

LIST OF TABLES: Table 1. Tier 1 Assays for o-Phenylphenol...........................................................8
Table 2. Estrogenic/Anti-Estroge nic Pathway for o-Phenylphenol..............................10
Table 3. Androgenic/Anti-Androgenic Pathway for o-Phenylphenol...........................14
Table 4. Thyroid Pathway for o-Phenylphenol.....................................................18

APPENDIX 1. EDSP Tier 1 Screening Assays..............................................................30
APPENDIX 2. Other Scientifically Relevant Information (OSRI)....................................42

APPENDIX 3. References Not Utilized in the o-Phenylphenol WoE Analysis......................55O-PHENYLPHENOL FINAL

iii Abbreviations

Abbreviation Terminology

A Androgen (hormonal pathway)

ADME Absorption, Distribution, Metabolism, Excretion

ALP Alkaline Phosphatase

ALT Alanine Aminotransferase

AMA Amphibian Metamorphosis Assay

ARTA Androgen Receptor Transcriptional Activation

AST Aspartate Aminotransferase

ANOVA Analysis of Variance
AOP Adverse Outcome Pathwa y A R Andro g en Rece p to r B max Bindin g at maximum BROD Benz y lox y resorufin-O-dealk y lase BUN Blood Urea Nitro g e n CA R Constitutive Androstane Rece p to r CFR Code of Federal Regulations

CG Cowper’s Gland

ChE Cholinesterase
ChEI Cholinesterase inhibition
CMC Carboxymethyl cellulose
CTA Comparative Thyroid Assay
CV Coefficient of Variation
CYP Cytochrome 450

DE R Data Evaluation Recor d DMSO Dimeth y l Sulfoxide DNA Deox y ribonucleic Aci d DO Dissolved Ox yg en DP Dorsolateral Prostrate
E Estro g en hormonal p athwa y EDSTAC Endocrine Disru p tor Screenin g and Testin g Advisor y Committee
EDRT Endocrine Disru p tor Review Team EDSP Endocrine Disru p tor Screenin g Pro g ram EE Ethin y l Estradiol ELISA Enz y me Linked Immunosorbent Assa y EOGRTS Extended One-Generation Re p roductive Toxicit y Stud y ( Rat )
E R Estro g en Rece p to r EROD Ethox y resorufin-O-dealk y lase ( or deeth y lase ) ERTA Estro g en Rece p tor Transcri p tional Activatio n EtOH Ethanol
F Female
F1 First filial g eneration
F2 Second filial g enerationO-PHENYLPHENOL FINAL

iv Abbreviation Terminology

Fcd Fecundit y FIFRA Federal Insecticide, Fungicide and Rodenticide Act
FOB Field Observation Batter y F Q PA Food Q ualit y Protection Act
Frt Fertilit y FSH Follicle Stimulatin g Hormone FSTRA Fish Short-Term Re p roduction Assa y FT Flutamide GD Gestation Da y GGT Gamma- g lutam y l Trans p e p tidase GnRH Gonadotro p in-releasin g hormone GP Glans Penis GSI Gonado-Somatic Index
H Hi g h
HLL Hind Limb Len g th HPG H yp othalamic-Pituitar y -Gonadal Axis HPLC/MS/MS Hi g h Pressure Li q uid Chromato g ra p h y /Mass S p ectrosco py HPT H yp othalamic-Pituitar y -Th y roidal Axis
I Inade q uate IC50 Inhibitor y Concentration at 50% of res p onse ICCVAM Intera g enc y Coordinatin g Committee on the Validation of Alternative K d Equilibrium Dissociation Constant

K OW Octanol/Water Partition Coefficient

L Low dose LABC Levator Ani-Bulbocavernosus LAGDA Larval Am p hibian Growth and Develo p ment Assa y LC50 Lethal Concentration in 50% of test organisms

LD Lactation Day

LH Luteinizing hormone

LOAEC Lowest Observed Adverse Effect Concentration

LOAEL Lowest Observed Adverse Effect Level
LOQ Limit of Quantitation

M Male
MDL Minimum Detection Level MEOGRT Medaka Extended One Generation Re p roduction Test
MH Medium hi g h
ML Medium low MoA Mode of Action MOE Mar g in of Ex p osure MRID Master Record Identifier

MROD Methoxyresorufin-O-dealkylase
MTC Maximum Tolerated Concentration
MTD Maximum Tolerated Dose
N Ne g ativeO-PHENYLPHENOL FINAL

v Abbreviation Terminology

NE Not examined/evaluate d
NF sta g e Nieuwkoo p and Faber’s Sta g in g Atlas NOAEC No Observed Adverse Effect Concentratio n NOAEL No Observed Adverse Effect Level NS Not Statisticall y Si g nificant N R Not Re p orte d OCSPP Office of Chemical Safet y Pollution and Prevention OECD Organization for Economic Co-Operation and Development

OPP Office of Pesticide Programs

ORD Office of Research and Develo p ment OSCP Office of Science Coordination and Polic y OSRI Other Scientificall y Relevant Informatio n P Positive
P Parental g eneratio n PC Positive Control PC 10 Positive Control at 10% of response

PC 50 Positive Control at 50% of response

PND Post-Natal Da y POD Point of De p arture PPS Pre p utial Se p aration PROD Pentaox y resorufin-O-dealk y lase ( or de p ent y lase )
PX R Pre g nane X rece p to r QC Quality Control

RBA Relative Binding Affinity

RBC Red Blood Cells
RfD Reference Dose RPC max Relative to Positive Control at maximum

SAP Scientific Advisor y Panel SC Solvent Control

s.c Subcutaneous SDH Sorbitol deh y dro g enase SDWA Safe Drinking Water Act

SEP Standard Evaluation Procedure SD Standard Deviation or Sprague-Dawley

SVL Snout-to-Vent Len g th SV Seminal Vesicles
T Th y roid ( hormonal p athwa y) T1WoERC EDSP Tier 1 Wei g ht of Evidence Review Committee T3 Triiodothyronine

T4 Thyroxine (tetraiodothyronine)

TP Testosterone Pro p ionate
T R Th y roid Rece p to r TSH Thyroid Stimulating HormoneO-PHENYLPHENOL FINAL

vi Abbreviation Terminology

UDPGT Uridine Diphosphate Glucuronyltr ansferase (also known as UGT)

VC Vehicle Control VO Vaginal Opening

VP Ventral Prostate VTG Vitello g eni n WoE Wei g ht-of-EvidenceO-PHENYLPHENOL FINAL

Page 1 of 56 Executive Summary

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether o-phenylphenol (o-PP) inte racts with E, A or T hormone pathways, the number and type of effects i nduced, the magnitude and patter n of responses observed across studies, taxa and sexes were considered. Additionally, the conditions under which effects occur were considered, in partic ular, whether or not endocrine-related responses occurred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On September 11, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) of the potential interaction of o-PP with the E, A or T hormone pathways. The T1 WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of the Tier 1 in

vitro assays followed by in vivo mammalian and wildlife results, then the results of the cited
OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

For o-PP, for the estrogen pathway, the Tier 1 in vitro estrogen receptor (ER) binding assay was equivocal results and positive for ER activa tion (Routledge and Sumpter, 1997 and Miller et al

., 2001). Additionally o-PP inhibited aromatase activ ity and increased estrad iol production in the
Tier 1 aromatase and steroi dogenesis assays, respectively. However, in the mammalian in vivo assays, there was no convincing evidence for an in teraction with the estrogen pathway. The uterotrophic assay was negative and in the female pubertal assay, the sole effect observed was a decrease in the percent regularly cycling animals at the high dose compared to the controls which occurred in the presence of syst emic toxicity. Due to the lack of redundancy of responses across the mammalian assays, there is insufficient evidence for a potential interaction in vivo in
mammals.

However, there are indications of potential effect s on the reproductive status in fish that included decreases in female vitellogenin, fecundity and fertility in the fi sh short-term reproduction assay (FSTRA); however, there were no effects on gonadal somatic index (GSI) or gonadal histopathology. In another fish reproduction study with the same species as the FSTRA, decreases in egg production were observed. These in vivo effects, along with indications ofO-PHENYLPHENOL FINAL

Page 2 of 56 potential in vitro effects including ER activ ation and altered steroidogene sis, suggest that o-PP may potentially interact with the estrogen pathway in fish.

For the androgen pathway, there was evid ence of potential interaction in the in vitro androgen receptor (AR) binding assay and conflicting evid ence in the steroidogenesis assay where a 30% increase in testosterone producti on at 10 μM o-PP and a 30% decr ease at 100 μM o-PP. Studies published in the open literature showed o-PP wa s not an androgen agonis t in the MDA-kb2 cell assay, however an anti-androgen effect of o-PP wa s observed with this cel l line. The Hershberger assay was negative and in the male pubertal as say, decreases seen in a number of androgendependent tissues were observed in the presence of overt toxicity. In the FSTRA, decreases in fertility were observed, however, no other androg en-related effects were observed in this study or in the OSRI fish reproduction study. There were no androgen-related effects observed in the Part 158 mammalian studies. Therefore, there is no convincing evidence fo r potential interaction with the androgen pathway.

For the thyroid pathway, there was no evidence of potential interaction of o-PP. In female pubertal assay, there were no treatment-r elated effects on thyroid hormones [T 4 and thyroid stimulating hormone (TSH)], thyr oid weights or thyroid histopa thology. In the male pubertal assays the only thyroid-related effect observed was a decrease in serum T 4 levels at all doses. In the amphibian metamorphosis assay (AMA), no tr eatment-related finding were observed except for a significant reduction in Day 21 normalized hindlimb length; however, given the lack of effects on developmental stage or thyroid histopathology, this finding alone is not considered to be sufficient evidence of a potential interaction of the thyroid pathway in amphibians. No thyroid effects were noted in any of th e Part 158 mammalian studies.

In summary, there is no convincing evidence of po tential interaction with the estrogen, androgen or thyroid pathways in vivo in mammals. There is also insufficient evidence of a potential interaction of the thyroid path way in amphibians. However, there is evidence of potential interaction with the estrogen pathway in non-mammalian species as suggested by the decreases in reproductive output in the FSTRA and fish OSRI. These findings in the fish assays along with
the results in the in vitro assays, suggest potential interaction with the es trogen pathway in fish. There is no convincing evidence of a potential interaction with the androgen pathway in fish.

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for o-PP since there was no convinci ng evidence of potential interaction with the estrogen, androgen or thyroid path ways. Effects on fecundity/fertility were observed at 876 μg
a.i./L in the FSTRA with eff ects on reproduction reported at 36 a nd 293 μg a.i./L in the fish OSRI assay (which was similar to the FSTRA in test design). Currently, there are no acceptable 40 CFR Part 158 chronic toxicity data for fish. At this time, based on the reproductive responses observed in the fish assays and the in vitro assays, EDSP Tier 2 Medaka Extended OneO-PHENYLPHENOL FINAL

Page 3 of 56 Generation Reproduction Test (MEGORT) is r ecommended to better understand potential reproductive effects in fish.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are not available for o-PP; however, were requested under the final workplan for the registration review for o-PP (USEPA 2014). The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consid ered sufficient for evaluating potential reproductive effects to birds from o-PP exposure. Relative to the EDSP, additional testing is not
recommended.O-PHENYLPHENOL FINAL

Page 4 of 56 I. Introduction

The Endocrine Disruptor Screenin g Programs (EDSP) Tier 1 a ssay battery is designed to provide the necessary empirical data to evaluate the potential of chemicals to interact with the estrogen (E), androgen (A) or thyr oid (T) signaling pathways. This interaction includes agonism and antagonism at the estrogen and androgen re ceptors, altered steroidogenesis, as well as hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituita ry thyroid (HPT) axes. In
addition to the available Tier 1 assay data, other scientifically relevant information (OSRI), including general toxicity data and open literature studies of sufficient quality were considered in this weight of evidence (WoE) assessment.

In determining whether O-phenylphenol (o -PP)interacts with E, A or T hormone pathways, the number and type of effect s induced, the magnitude and pattern of responses observed across studies, taxa and sexes were considere d. Additionally, the conditions under which effects occur were considered, in particular, whether or not endocrine-related responses occu rred at dose(s) that also resulted in general systemic toxicity or overt toxicity.

On September 11, 2014, the EDSP Tier 1 Assa y Weight of Evidence Review Committee (T1WoERC) of the Office of Pesticide Programs (OPP) and the Office of Science Coordination and Policy (OSCP) conducted a we ight-of-evidence (WoE) analysis of the potential interaction of o-PP with the E, A or T hormone pathwa ys. The T1WoERC conclusions from the WoE evaluation in this report are presented by pathway (E, A and then T) beginning with the results of
the Tier 1 in vitro assays followed by in vivo mammalian and wildlife results, then the results of
the cited OSRI for mammalian and wildlife studies (40 CFR Part 158 and literature).

Ortho-phenylphenol (o-PP) has a water solubility of 700 mg/L. Due to a vapor pressure of

2×10

− 3 mm Hg at 25°C, o-PP is anticipated to exis t solely as a vapor if released to the atmosphere. The octanol/water partition coefficient (log K ow ) is 3.3 and a bioconcentration factor of 51 in fish. o-PP is stable and persistent in abiotic aqueous medium, but is not stable photolytically. It is immobile in soils, but has a potential for vol atilization from mo ist soils.

The available information considered in determin ing the potential interacti on of o-PP with the E, A, or T pathways include submitted EDSP Tier 1 assays and/or other scientifically relevant information (OSRI) such as general toxicity stud ies and other published articles. These data are summarized in Sections III.A through III.C. An analysis of the data submitted to the agency, using the WoE approach outlined by the Agency (U SEPA, 2011), is presented in Section IV. The EDSP Tier 2 study recommendations ar e presented in Section V.O-PHENYLPHENOL FINAL

Page 5 of 56 II. Sources of Scientific Data and Technical Information

A. EDSP Tier 1 Screening Assays

The Tier 1 assays and/or other scientifically relevant information (OSRI) submitted to satisfy the agency’s test order are shown below in Table 1. Executive Summaries are presented in Appendix

1. B. Other Scientifically Relevant Information (OSRI)

In response to the Agency’s Test Orders, data believe d to be relevant to one or more of the Tier 1 assays were submitted as OSRI by the Test Order recipients and/or the public. This included studies published in the open literature and/or data submitted to support pesticide registration ( e.g. , Part 158 Guideline studies). The Agency’s re view of the initial OSRI is provided in the Report of the Endocrine Disruptor Review T eam on o-PP (USEPA, 2010). Since then, the Agency has also conducted a more recent search of available scientific literature for any
additional relevant information. Summaries of the available OSRI are presented in Appendix 2. Additionally, literature/studies c onsidered but not util ized for the WoE analysis are listed in Appendix 3.

III. Weight of Evidence (WoE) Evaluation

The principles, criteria and approach used in the WoE determination on the potential of a
substance to interact with endocrine-related processes ( i.e., E, A or T hormone pathways) were as described in the WoE guidance document (USEPA, 2011) and presen ted at the 2013 FIFRA Scientific Advisory Panel (SAP) (USEPA, 2013). The weight of evidence process identifies how
the individual lines of evidence are asse mbled and integrated along two concepts ( i.e

., complementarity and redundancy) within the co nceptual framework of an adverse outcome pathway). Broadly, there are four main st eps outlined in the guidance which provide the foundation for WoE evaluations. The first step is to evaluate the indivi dual studies for their scientific quality and relevance in evaluating potential endocrine interaction(s). The second step is to integrate the data along different levels of biological organization while examining the extent of complementarity ( i.e ., the concordance of endpoints within an assay that measures multiple endpoints) and redundancy ( i.e ., the concordance of endpoint s/responses across assays) in the observed responses across these different le vels of biological orga nization. The third step
is to characterize the main lines of evidence as well any conclusions. Finally, the last step is to
evaluate whether additional te sting is needed based on the evidence and conclusions described
above.

As mentioned, the first step is to assemble and evaluate the available scientific data. Data for the
EDSP Tier 1 WoE evaluation falls into one of two categories: 1) EDSP Tier 1 data, or 2) other scientifically relevant information (OSRI). The EDSP Tier 1 data include a battery of 11 assays consisting of in vitro and mammalian and wildlife in vivo assays. The Tier 1 assays wereO-PHENYLPHENOL FINAL

Page 6 of 56 designed specifically to evaluate a number of key biological events including potential effects on receptor binding (estrogen and androgen agonist and antagonist), steroidogenesis, and other effects on the HPG and HPT axes. OSRI may in clude published literature studies as well as studies conducted under USEPA (o ften referred to as Part 158 data) or OECD guidelines submitted in support of pesticide registrations. E ach study is evaluated for scientific quality and relevance for informing interactions with the E, A or T pathway. Additionally, the consistency of the responses in the individual study is evaluated. For the Tier 1 in vivo assays, often multiple endpoints are measured in each assay.

Evaluation of the potential confounding effects of overt toxicity in the study as well as the relative degree of diagnostic utility of a speci fic endpoint for discerni ng whether or not the chemical has interacted with the endocrine syst em, are considered. The co llective response of the individual endpoints, as well as the conditions under which they were expressed, are considered when evaluating an overall indi cation of potential interaction as measured by the study.

The second step in this WoE process is to form ulate hypotheses and integr ate the available data along different levels of biological organization. Two keys elements in the integration of data, as well as characterizing the extent to which the available data suppor t a hypothesis that a chemical has the potential to interact E, A or T path ways, are the concepts of complementarity and redundancy. These two concepts provide a basis for considering the plau sibility, coherence, strength, and consistency of the body of evidence. The current EDSP Tier 1 screening assays are
meant to evaluate whether or not a chemical can interact with E, A and T consisting of different levels of biological organization from a molecular initiating ev ent, such as receptor binding, through potential adverse effects in apical endpoint s such as sexual develo pment and fecundity at the whole organism level. The extent of expression of responses at higher levels of biological organization can indirectly provide information on potential compensatory capabilities of an individual organism.

After the data have been assembled and integrated, the third step is to characterize the main lines of evidence along with the conclusions; this characterization involves three components. The first component is whether the data provide rele vant, robust and consistent evidence in terms of complementarity and redundancy as well as biologi cal plausibility. Second, is at what level of biological organization were the responses observed and whether organisms exhibit compensatory responses at higher levels of bi ological organization? Finally, under what conditions did the responses occur including co nsideration of whether the responses were observed in the presence of overt or systemic toxicity? The presence of overt and/or systemic toxicity introduces uncertainty in the ability to distinguish eff ects specifically related to an
endocrine-related effect from a non-endocrine toxic response.

This uncertainty in distinguishi ng whether the responses were endoc rine-related was discussed at the FIFRA SAP meeting that evaluated scientific issues associated with the WoE evaluation of the EDSP Tier 1 screening process. In October 2013, the SAP stated that , “ In summary, theO-PHENYLPHENOL FINAL

Page 7 of 56 Panel agreed that little, if any, we ight should be placed on signs of endocrine disruption in the presence of overt toxicity. All e ffects in endocrine sensitive tissues should be evaluated in terms of primary interactions with the endocrine system vs. secondary effects rela ted to toxicity in nonendocrine organs or overall disruptions in homeostasis” (USEPA, 2013).

For these WoE analyses, overt toxicity was ge nerally defined in accordance with EPA’s current approach as used by o-PP in reviewing 40 CF R Part 158 studies for bot h human and ecological risk assessments. Specifically, in these analyses, the effects that EPA considered to be potential evidence of overt toxicity include d, but were not limited to: mortal ity; clinical signs such as tremors, ataxia and abnormal swimming (fish and aquatic-phase amphibians); and body weight
decreases of ≥ 10% in mammals. Additionally, other effects including morphological ( e.g ., organ weights/histopathology), biochemical ( e.g ., blood chemistry), and other clinical signs ( e.g ., lethargy) were also considered when evaluating overt toxicity, es pecially if the effects were extreme. In some instances, one parameter ( i.e ., death or >10% decrease in mammalian body weight) was sufficient to consider a dose/concentra tion to be overtly toxic. However, in other instances, more than one parameter was needed to determine overt toxicity. For example, in the FSTRA, generally, body weight decr eases were considered along with other responses when assessing potential overt toxicity. Additionally, effects which were considered to be signs of systemic toxicity were also captured and these ef fects were generally cons idered as less severe forms of toxicity ( e.g ., changes in organ weights or blood chemistry). The circumstances for
which a dose/concentration was co nsidered overtly toxic for a pa rticular study are described in
Section IV.A.

In summary, EPA considers multiple lines of evidence in including the observed responses in the Tier 1 assays and OSRI in the context of a chemical’s physical/chemi cal properties and its
known modes of action in its overal l characterization of a chemical ’s potential to interact with the E, A or T pathway. Adequately addressing the three main components described above is fundamental to the WoE process and in determ ining whether additional data are needed. In addition to characterizing the WoE, reviewers also considered: 1) uncertainties and their potential impact to conclusions; 2) discussion of key studies; 3) description of inconsistent or conflicting data; 4) overall strength of eviden ce supporting a conclusion; and, 5) what, if any, additional data are needed and why. Assessing th e need for additional data is based on a caseby-case analysis which took all availabl e toxicity data into account.

The WoE approach involved consideration of data (i.e., lines of evidence) from the EDSP Tier 1 assays and OSRI which are depicted in Tables 2 - 4

. These tables contain data that are considered scientifically and bi ologically relevant with regard to a treatment-related effect which supports a conclusion of whether or not a s ubstance has the potential to interact with the E, A, or T pathway. Effects that occurred in the presence of overt toxicity are discussed in the text for each respective pathway (E, A or T) but ar e not reported in the table for E, A or T.O-PHENYLPHENOL FINAL

Page 8 of 56 A. EDSP Tier 1 Screening Assays

The Tier 1 assays submitted in response to the agency’s test order for o-PP are shown below in

Table 1

. Table 1. Tier 1 Screening Assays for o -Phenylphenol.

Tier 1 Assays Test Guideline Test Order Status

ER Binding Assay (Rat uterine cytosol) OCSPP 890.1250 Requirement Satisfied (MRID No. 48672604))

ER α Transcriptional Activation Assay (Human cell line HeLa 9903) OCSPP 890.1300; OECD 455 Requirement Satisfied by OSRI (Routledge and Sumpter, 1997 and Miller et al ., 2001)

AR Binding Assay (Rat prostate cytosol) OCSPP 890.1150 Requirement Satisfied (MRID No. 48672603)

Steroidogenesis Assay (Human cell line H295R) OCSPP 890.1550; OECD 456 Requirement Satisfied (MRID No. 48672601)

Aromatase Assay (Human recombinant microsomes) OCSPP 890.1200 Requirement Satisfied (MRID No. 48672608)

Uterotrophic Assay (Rat) OCSPP 890.1600; OECD 440 Requirement Satisfied (MRID No. 48672605)

Hershberger Assay (Rat) OCSPP 890.1400; OECD 441 Requirement Satisfied (MRID No. 48672602)

Pubertal Female Assay (Rat) OCSPP 890.1450 Requirement Satisfied (MRID No. 48686601)

Pubertal Male Assay (Rat) OCSPP 890.1500 Requirement Satisfied (MRID No. 48686602)

Fish Short-term Reproduction Assay OCSPP 890.1350; OECD 229 Requirement Satisfied (MRID No. 48672606)

Amphibian Metamorphosis Assay (Frog) OCSPP 890.1100; OECD 231 Requirement Satisfied (MRID No. 48916901)O-PHENYLPHENOL FINAL

Page 9 of 56 B. Effects on Hypothalamic-P ituitary-Gonadal (HPG) Axis

1. Effects on Estrogen Pathway

The potential for o-PP to interact with the estrogen pathway is summarized in Table 2. The various targets of the estrogen pathway across the relevant Tier 1 a ssays are delineated so as to facilitate determination of potential for estrogenic, anti-estrogenic or HPG axis effects. This table also includes HPG-relevant findings from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the table and not considered further in the WOE assessment.

In the Tier 1 in vitro assays, aromatase activity was inhibi ted in a dose-dependent manner at 10

− 4

and 10

− 3 M in the aromatase assay, and dose-depende nt induction of estradiol production was observed in the steroidogenesis assay at concentrations of 10 -5 and 10 -4 M (2.6- and 4.0-fold, respectively).

In the Tier 1 binding assay, o-PP appeared to disp lace estradiol from the ER in a concentrationdependent manner in all three runs, however the binding curve exhibited steep Hill slopes, suggesting some other type of interference inst ead of direct competition for ER binding. Blair et
al ., 2000 reported negative results for ER binding a ssay in rat uterine cytosol. In OSRI used to satisfy the Tier 1 ERTA assay, no estrogenic activ ity was seen in ER-related transcription in yeast expressing the human ER α gene (Routledge and Sumpter, 1997 and Miller et al., 2001).

o-PP was negative in the Tier 1 in vivo uterotrophic assay. In the female pubertal rat assay, the percent regularly cycling animals in the high- dose (900 mg/kg/day) was decreased (28.6% vs. 86.7% controls), and there was a dose-dependent tendency to spend more than 4 consecutive days in diestrus (1/15 control, 2/15 low, 3/ 15 mid, 7/15 high dose), leading to 4 animals not cycling ( i.e . spending all the time in diestrus) at th e high dose. The cyclicity effects were observed in the presence of syst emic toxicity including kidney hist ological changes and increases in serum urea nitrogen. There we re no other estrogen-related eff ects in this study. There were no estrogen-related effects observed in the Part 158 mammalian toxic ity studies. In a study by Kaneda, 1978, fetal resorptions were increased at a dose that was overtly toxi c (600 mg/kg/day).

In the FSTRA, while fecundity or fertility were not significantly (p>0.05) decreased at the high concentration (876 μg a.i. /L; ↓ 47.5 and 32%, respectively), they were considered to be biologically meaningful decreases. Additionally, female VTG concentration also was decreased (p<0.05; 35.5%) at the high-trea tment level. There were no other treatment-related effects observed. In an OSRI, non-guideline fish reprodu ction study with fathead minnow (which was similar to the FSTRA), while c ontrol fecundity was observed to be variable in the study, data suggested a decrease in fecundity (total numbe r of eggs per female) at 36 and 293 μg/L. There were no treatment-related effects on GSI, VT G induction, or growth (length and weight). Gonadal histology or plasma sex steroids were no t analyzed in this study (reportedly due to no evidence of VTG induction).O-PHENYLPHENOLFINALPage 10 of 56Table 2. Estrogenic/Anti-Estrogenic Pathway foro-PhenylphenolLines of Evidence Indicating Potential Interactionwith the Estrogenic/Anti-Estrogenic Pathway foro-Phenylphenol1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysER Binding(MRID 48672604)EERTARequirement satisfied by OSRI (Routledge and Sumpter, 1997, and Milleret al., 2001; see below)Aromatase(MRID 48672608)PSteroidogenesis(MRID 48672601)PUterotrophic(MRID 48672605)NN NFemale Pubertal Rat(MRID 48686601)NNNNP(H)4NLW:↓9%ADN:↓13%BUN:↑23%ALT:↑2XKidney: Hist(H)NFSTRA(MRID 48672606)NEN NNP5↓35.5%(H)N6OSRIER Binding (Blairetal., 2000)NERTA (Routledgeand Sumpter, 1997)PO-PHENYLPHENOLFINALPage 11 of 56Table 2. Estrogenic/Anti-Estrogenic Pathway foro-PhenylphenolLines of Evidence Indicating Potential Interactionwith the Estrogenic/Anti-Estrogenic Pathway foro-Phenylphenol1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3ERTA (Milleret al.,2001)PSubchronic toxicity(Dog; MRID41656401)NE NE N NEX(M, H)NDevelopmentaltoxicity (Rat; Kanedaet al., 1978)NX(ML, MH,H)X(MH,H)Developmentaltoxicity (Rat; MRID00164362)NENX(H)NDevelopmentaltoxicity (Rabbit;MRID 41925003)NNNX(H)Two-generationreproduction (Rat;MRID 41729401)NE N NNNXX(H)Two-generationreproduction (Rat;MRID 43928801)NE N NNNXX(H)Chronic toxicity/carcinogenicity (Rat;MRID 43954301)NENN NEX(L, M, H)X(H)Carcinogenicity(Mouse; MRID43545501)NE NE N NEX(M, H)X(H)O-PHENYLPHENOLFINALPage 12 of 56Table 2. Estrogenic/Anti-Estrogenic Pathway foro-PhenylphenolLines of Evidence Indicating Potential Interactionwith the Estrogenic/Anti-Estrogenic Pathway foro-Phenylphenol1Study Type/Literature Citation ER Binding ER Activation Steroidogenesis Sex Steroid Hormone Uterine Weight Ovarian Weight/GSI Ovarian/Gonad Staging and Histopathology Pituitary Weight Estrous Cyclicity Age & Weight at VO 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Chronic toxicity(Dog; MRID41656401)NENNNX(M, H)NFreshwater fishreproduction (Fatheadminnow; MRID48136404)N NENEP(MH, H)7NN1.Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatmentArrows (↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. LW: Liver weight; ADN: Adrenal; BUN: Blood Urea Nitrogen; ALT: Alkaline transferase;Histo: Histopathology.2. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X inthis column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. The percent of females that were regularly cycling was decreased from 86.7% to 28.6% in the high-dose group.5. Fertility success was decreased 32% at the high-dose and fecundity was decreased 47%; while not significantly different (p>0.05), it is considered to bebiologically significant.6. While survival was 79% in the highest treatment concentration(876 μg a.i./L), one test vessel was observed to have 100% mortality, accounting for 6 of the7 total mortalities at this concentration which may indicate atank-related issue and not treatment-related mortality. There were no other observed clinicalsigns of toxicity or significant effects on body weight or length.7. Decreased egg production (total number of eggs per female) at the mid-high and high concentration.E EquivocalP Positive findings (in vitro)/No effect (in vivo)N Negative findings (in vitro)/No effect (in vivo)NE Not examinedO-PHENYLPHENOL FINAL

Page 13 of 56 2. Effects on Androgen Pathway

The potential for o-PP to interact with the androgen pathway is summarized in Table 3. The various targets of the androgen pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for androgenic, anti-androgenic or HPG axis effects. This
table also includes HPG-rele vant findings from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are di scussed in the text but are not reported in the table and not considered furthe r in the WOE assessment.

o-PP was positive in the Tier 1 in vitro androgen receptor (AR) binding assay with an estimated mean Log IC 50 of

− 3.749 Log M. In a published AR transc riptional activati on (ARTA) study by

Orton et al . (2011), o-PP was not an androgen agonist in the M DA-kb2 cell assay, however an anti-androgen effect of o-PP wa s observed in this assay (IC 20 = 3.43 μM). The Tier 1 steroidogenesis assay showed conflicting results with a 30% increase in testosterone production at 10 μM, and a 30% decrease at 100 μM.

o-PP was negative in the Tier 1 in vivo Hershberger assay. In th e male pubertal assay, no
treatment-related effects were observed on serum testosterone concentrations. The adjusted age of attainment of preputial separation (PPS) was increased by 1.6 days in the high-dose group (45.1 vs. 43.5 days control), but this change from control was not statistically significant. Numerous androgen-dependent ti ssues had decreased mean weights at the high dose when compared to corresponding controls. Unadjusted and adjusted pituitary weights were decreased in the high-dose animals (15% and 10%, respectively). All these effects, however, were seen only in the presence of overt toxi city that manifested as decrea ses in body weight and systemic effects including increases in li ver and adrenal weights, increas ed serum blood urea nitrogen and histopathological lesions of th e kidneys. The only androgen-relate d effect observed in the Part 158 mammalian toxicity studies was an increase in absolute (34%) and relative (46%) testes weights observed in high-dose males in the chronic toxicity study in rats, how ever this effect cooccurred with 10-12% decreased body weight (compared to controls ) throughout the exposure time and 11% decreased body weights at termination.

In the FSTRA, fertility was decreased 32% at th e high treatment level, however, there were no other effects observed in male fish. Additionally , there were no male effects noted in the nonguideline freshwater fi sh reproduction study.O-PHENYLPHENOLFINALPage 14 of 56Table 3. Androgenic/Anti-Androgenic Pathway foro-PhenylphenolLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway foro-Phenylphenol1Study Type/Literature Citation AR Binding/Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3EDSP Tier 1 AssaysAR Binding (MRID48672603)PSteroidogenesis(MRID 48672601)P4Hershberger (MRID48672602)NX(H);NMale Pubertal Rat(MRID 48686602)NNNNNNNNLW:↑21%;ADN:↓16%BUN:↑27%↓12%BW(H)FSTRA(MRID 48672606)N NNFrt:(H)5NN6OSRIARTA (Ortonet al.,2011)PSubchronic toxicity(Dog; MRID41656401)NE N NE N NE NEX(M, H)NDominant Lethal(Mouse; Kanedaetal., 1978)NN NO-PHENYLPHENOLFINALPage 15 of 56Table 3. Androgenic/Anti-Androgenic Pathway foro-PhenylphenolLines of Evidence Indicating Potential Interaction with the Androgenic/Anti-Androgenic Pathway foro-Phenylphenol1Study Type/Literature Citation AR Binding/Activation Steroidogenesis Sex Steroid Hormones Testes Weight/GSI Gonad Staging and Histopathology Epididymides Weight Epididymides Histopathology Pituitary Weight Accessory Sex Organ Weights/ 2° Sex Characteristics Fertility(Frt) /Fecundity (Fcd) Age and Weight at PPS Vitellogenin (VTG) Systemic Toxicity Observed 2 Overt Toxicity Observed 3Two-generationreproduction (Rat;MRID 41729401)NNNENNNX(H)Two-generationreproduction (Rat;MRID 43928801)N NNENNX(H)X(H)Chronic toxicity/carcinogenicity (Rat;MRID 43954301)N NNE NNEX(H)X(H)Carcinogenicity(Mouse; MRID43545501)NNNENNEX:(L, M, H)X(H)Chronic toxicity(Dog; MRID41656401)N NNE N NX(M, H)NFreshwater fishreproduction (Fatheadminnow; MRID48136404)NNENENNN1. Key to responses: L=Low treatment, ML=Medium-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows(↓or↑) indicate the direction of the response. Ashaded cell indicates that is parameter is not routinely evaluated or is not applicable in this assay.Changes in weight are absolute unless otherwise indicated. Abbreviations for androgen sensitive tissues: Seminal vesicles (SV), Ventral prostate (VP),Dorsal prostate (DP), Prostate (PR), Levator ani-bulbocavernosus (LABC), Epididymides (E), Cowper’s gland (CG), glans penis (GP). BW: Bodyweight; LW: Liver weight; ADN: Adrenal weight; BUN: Blood Urea Nitrogen; Histo: HistopathologyO-PHENYLPHENOLFINALPage 16 of 562. The systemic toxicity in the Tier 1 assays are presented in this column (e.g. KW= kidney weight). The systemic toxicity for the OSRI is indicated by anX in this column. For details see Section IV. A3. The overt toxicity in the Tier 1assays are presentedin this column (e.g.↓BW). The overt toxicity for the OSRI is indicated by an X in this column. Fordetails see Section IV. A4. A 30% increase in testosterone production was observedat 10 μM o-PP; however a30% decrease at 100 μM o-PP5. Fertility success was decreased 32% atthe high-dose and was not statistically significant but was considered biologicallymeaningful.6. While survival was 79% in the highest treatment concentration, one test vesselwas observed to have 100% mortality, accounting for 6 of the 7 totalmortalities at this concentration which may indicate a tank-related issue and not a treatment effect on mortality. There wereno other observed clinicalsigns of toxicity or significant effects on body weight or length.P Positive findingsN Negative findings (in vitro)/No effect (in vivo)NE Not examinedO-PHENYLPHENOL FINAL

Page 17 of 56 C. Effects on Hypothalamic-Pit uitary-Thyroidal (HPT) Axis

The current EDSP Tier 1 batte ry does not have a specific in vitro assay to detect chemicals with the potential to affect hypothalamic or pituitary regulation of thyroid hormone production, but it
does include three in vivo assays that provide data to de tect changes in the HPT axis, i.e ., the pubertal female and male rat assays, and the AMA (frog).

The potential for o-PP to interact w ith thyroid regulati on are summarized in Table 4. The various targets of the th yroid pathway across the relevant Tier 1 assays are delineated so as to facilitate determination of potential for thyroid or HPT axis effects. This table also includes HPG-relevant findings from the data evaluated as OSRI. Effects that occurred in the presence of overt toxicity are discussed in the text but ar e not reported in the ta ble and not considered further in the WOE assessment.

No effects on thyroid weight or histopathology were observed in the in vivo male and female pubertal assays. TSH concentrations were not affected, but serum T4 concentrations were decreased in all treatment groups in the male pubertal assay; however the T4 changes at the highest dose males occurred in th e presence of over t toxicity.

In the AMA, o-PP did not alter the median Nieu wkoop-Faber (NF) developmental stage at Day 7 or 21 relative to the negative control. Th ere were no treatment related effects on thyroid histopathology. There were no signif icant (p>0.05) effects on Day 7 normalized hind-limb length (HLL), but a significant decrease of 8% was observed in HLL on Day 21 at the high test
concentration. There were also no significant (p>0.05) differences in snout-to-vent length (SVL) and body weight.

No thyroid effects were noted in any of the Part 158 mammalian studies.O-PHENYLPHENOL FINAL

Page 18 of 56 Table 4. Thyroid Pathway for o-Phenylphenol

Lines of Evidence Indicating Potential Interact ion with the Thyroid Pathway for o-Phenylphenol 1

Study Type/ Literature CitationThyroid WeightThyroid: Gross andHistopathologySerum T4LevelsSerum TSH LevelsPituitary WeightDevelopmental stage(± or asynchronous,HLL)Growth (BW, BL,SVL)Systemic ToxicityObserved2Overt ToxicityObserved3

EDSP Tier 1 Assays

Female Pubertal Rat (MRID 48686601) N N N N N LW: ↓ 9% ADN: ↓ 13%
BUN: ↑ 23% ALT: ↑ 2X Kidney: Histo (H) N

Male Pubertal Rat (MRID 48686602 ) N N ↓ 15, 23% (L, M) N N LW: ↑ 21%; ADN: ↑ 16%
BUN: ↑ 24% Kidney: Histo (H) ↓ 12%
BW (H)

AMA (Frog; MRID 48916901) N Day 21
HLL: ↓ 8% (H) N N

OSRI Subchronic toxicity (Dog; MRID 41656401) NR N NE X (M, H) N
Two-generation reproduction (Rat; MRID 41729401) NE NE NE X X (H)

Two-generation reproduction (Rat; MRID 43928801) NE NE NE X (H) X (H)

Chronic toxicity/ carcinogenicity (Rat; MRID 43954301) N N NE X X (H)

Carcinogenicity (Mouse; MRID 43545501) NE N NE X X (L, M,

H ) Chronic toxicity (Dog; MRID 41656401) N N N X (M, H) N
1. Key to responses: L=Low treatment, ML=Mediu m-low treatment, M=Medium treatment, MH=Medium-high treatment, H=High treatment. Arrows ( ↓ or ↑ ) indicate the direction of the response. A shaded cell indicates that is parameter is not routinely evaluated or is not app licable in this assay. Changes in weight are absolute unless otherwise indicated. BW: Body weight; LW: Live r weight; ADN: Adrenal weight; BUN: Blood Urea Nitrogen; Histo: Histopathology. 2. The systemic toxicity in the Tier 1 assays are presented in this column ( e.g . KW= kidney weight). The systemic toxicity for the OSRI is indicated by an X in this column. For details see Section IV. AO-PHENYLPHENOL FINAL

Page 19 of 56 3. The overt toxicity in the Tier 1 assays are presented in this column ( e.g

. ↓ BW). The overt toxicity for the OSRI is indicated by an X in this column. For details see Section IV. A N Negative findings NE Not examined NR Not reportedO-PHENYLPHENOL FINAL

Page 20 of 56 IV. Committee’s Assessment of Weight of Evidence

This section of the document describes the we ight of evidence (WoE ) determination on the potential of o-PP to interact w ith endocrine related processes ( i.e., E, A, or T hormonal pathways) as well as recommendati ons regarding Tier 2 testing.. The results of the Tier 1 assays are considered, along with other scientifically relevant information ( e.g., 40 CFR Part 158 test guidelines and published or publicly available pe er-reviewed studies). WoE analysis in the context of the EDSP follows the Agency’s gui dance (USEPA 2011) and is conducted on a caseby-case basis by first assessing the different lines of evidence ( i.e., specific Tier 1 assays and OSRI), then performing an integrated an alysis of those line s of evidence.

The WoE evaluation includes consid erations of biological plausibility of the findings from the different lines of evidence by examining the co nsistency, coherence, and interrelationships among the measured endpoints within and across studies. The available findings from standard toxicology studies on the substance may contribute to the WoE evaluati on in helping el ucidate if effects seen in the Tier 1 assay are relate d to perturbations of the endocrine system per se or alternatively a sequelae of system ic effects. Endocrine modes of action may elicit a number of phenotypic consequences other than thos e evaluated in the Tier 1 assays.

Endocrine-related findings in the presence of overt toxicity may result in uncertainty as to whether or not the responses ar e related through an endocrine pathway, therefore non-endocrine toxic responses (including but not limited to mortality or body weight changes) are also
considered in this WoE evaluation. The FIFRA SAP that evaluated scientific issues associated with weight of evidence eval uation of the results of the Tier 1 assays stated that “In summary, the Panel agreed that little, if any, weight shoul d be placed on signs of endocrine disruption in the presence of overt toxicity. A ll effects in endocrine sensitive tissues shou ld be evaluated in terms of primary interactions with the endocrine system vs. secondary effect s related to toxicity in non-endocrine organs or overal l disruptions in homeostasis” (USEPA, 2013).

A. Systemic/Overt Toxicity in the in vivo Tier 1 Assays and OSRI

Effects that were considered to be systemic or overt toxicity for the in vivo Tier 1 assays and
OSRI studies are described belo w. Generally, one parameter (i .e., death or >10% decrease in mammalian body weight) was sufficient for a dose/concen tration to be considered overtly toxic. However, in other instances, more than one para meter was needed to determine overt toxicity. Effects which were considered to be signs of sy stemic toxicity were ge nerally less severe forms of toxicity (e.g., changes in orga n weights or blood chemistry).O-PHENYLPHENOL FINAL

Page 21 of 56 1. Tier 1 in vivo Assays

In the Hershberger assay, the majority of high-dose (1000 mg/kg/day) animals (6/7 in the androgenic portion and 5/7 in the anti-androgenic portion) displayed some or all of the following clinical signs: clear or red perioral soiling, pe rineal soiling (urine and/or feces), decreased activity, noisy respiration, and soft feces. At necropsy, one animal in each high-dose group (androgenic and anti-androgenic portions) was found to have th ickening of the non-glandular mucosa of the stomach. Adrenal, liver and kidney weights of treatment groups at necropsy were comparable to the control group.

In the androgen agonist portion of the Hershbe rger assay, mean body weight was decreased by 15% on Day 7 in the high-dose group; however, mean body weights at the other determinations were only slightly decreased (NS; ↓ 6-8%). Food consumption in this group also was decreased by approximately 30-40% between Days 1-7, but was comparable to the negative control during Days 7-11. Body weights and food consumption in the lower dose groups were comparable to controls throughout the du ration of the study.

In the anti-androgen portion of the Hershberger assay, body weights in o-PP-treatment groups were generally comparable to the control group; mean food consumption was decreased 20% in the high-dose animals, with no trea tment effects at the lower doses.

In the uterotrophic assay, body weights in the treated groups were comparable to control throughout the duration of the study. The onl y organ assessed was the uterus, therefore additional information on systemic toxici ty for this assay is not available.

In the female pubertal rat assay, mean body we ights generally were similar across all dose groups at all times measured. At the high dose ( 900 mg/kg/day), relative ( but not absolute) liver weight was increased 9%, and adjusted (but not absolute) adrenal wei ghts were decreased 13%; histological analysis of this or gans is not available. Kidney we ights of treated groups were not different from the control group, however the high dose group had elevated serum nitrogen urea levels (23%) compared to controls and histol ogical examination found increased incidence of collecting duct epithelium hypertrophy (0/15 co ntrol, low and mid dose vs. 3/14 high dose), multifocal tubule degeneration (4/15 control, 9/15 low and mid, 12/14 high), multifocal tubule dilation (1/15 control, 0/15 low, 1/15 mid, 9/14 high), a nd focal tubule necrosis with inflammation (0/15 controls, low an d mid doses vs. 2/14 high dose).

In the male pubertal assay, mean terminal body weight was decreased by 12% in the high-dose group (900 mg/kg/day); body weights were not affected in the lo wer dose groups. Mean relative and adjusted (but not absolute) liver weights were increased (21% and 10%, respectively) at the high dose. Mean absolute adrenal weights were decreased at the low, mid and high doses (11, 11 and 16%, respectively), but relati ve and adjusted weights were not different from controls. Kidney weights of treated groups were not different from the control group, however the highO-PHENYLPHENOL FINAL

Page 22 of 56 dose group had elevated serum nitrogen urea levels (27%) compared to cont rols and histological examination found increased incidence of collecting duct epitheliu m hypertrophy (0/15, 1/15, 0/15 and 5/14) and multifocal tubule dilation (1/1 5, 0/15, 2/15, 11/14); there was also increased severity (but not increased incidence) of multifocal tubule degeneration (very slight vs. slight).

In the FSTRA, there were no clinical signs of toxicity or significant (p>0.05) effect on body weight or length. In the highest treatment concentration (0.876 mg a.i/L, mean measured time weighted average), mortality was by 31% in the high-dose females and was 28% overall across both genders. One test vessel was observed to have 100% mortality, acc ounting for 6 of the 7
total mortalities at this concentration. It was reported that these mortalities may have been tank
related and not treatment related since other tanks in this treatment concentration did not have similar results. In the AMA, there were no tr eatment-related effects on survival [(only a single mortality was observed at the highest treatmen t concentration (1.92 mg a.i/L, mean measured time weighted average)] and there were no notab le clinical signs of toxicity reported.

2. OSRI

In a subchronic toxicity study in dogs, dose-relate d emesis was noted in a ll treated animals with the high-dose group exhibiting the most severity. Body weight gains in all study animals were variable, but treated animals gene rally gained less (or lost) we ight during the study period. In a developmental toxicity study in rats, there were no reported clinical observations related to treatment. Body weight gains during GDs 6- 16 were decreased in the medium- and the highdose groups, with slight overall body weight gain (GDs 6-21) d ecreases in both groups. In a developmental toxicity study in rabbits, ten deaths occurred during the stu dy. At least four of these deaths, in the high-dose group, were consider ed to be treatment-related, and an abortion at the high dose also may have been related to treatm ent. Clinical signs (incidences of decreased feces and perineal soiling) may have been related to treatment in the medium-dose, as well as the high-dose, group.

In a two-generation reproduction study in rats, ther e were no treatment-rela ted clinical signs of toxicity in any treated groups (adults and pups), and no treatm ent-related increase in mortality, although the death of one medium-dose animal pr obably was due to potentia lly treatment-related kidney failure. Body weights were decreased ( ↓ 6-13%) during the pre-mating periods in highdose F0 and F1 parents, as well as during the rest period between the F1a and F1b, and the F2a and F2b, breedings. Although body weights were o ccasionally decreased in females during the gestation and lactation periods , these decreases were genera lly <10% and body weight gains were not affected. Food consumption also wa s decreased in the high-dose F0 and F1 groups during the rest periods between breedings.

In another two-generation repr oduction study in rats, one P gene ration high-dose male died due to treatment-related kidney failure . Additionally at th is dose, an increase in the incidence of urine staining was noted in the P and F1 generation males. Th is finding was associated withO-PHENYLPHENOL FINAL

Page 23 of 56 treatment-related gross and micros copic lesions of the urinary trac t in these animals. Decreased body weights during pre-mating were observed in high-dose P females, but not males, with greater decreases (generally ≥ 10%) in high-dose F1 males and fe males. Body weight decreases continued in the females during gestation and lactati on, but the decreases were generally <10%.

In the chronic toxicity/carcinogenicity study in rats, there was no treatment-related effect on mortality observed. There was an increase in abnormal color urination and red staining in highdose males (402 mg/kg/day), brown staining in medium- and high-dose females (248 and 647 mg/kg/day), and urine staining in high-dose males and all treated females. Slight body weight
decreases were noted in the medium-dose an imals in the two-year oncogenicity group (no decrease in the one-year in terim sacrifice group) at Weeks 13, 52, and 78, with weights comparable to control by Week 104. Body wei ght gains in the medium-dose animals were generally >90% of control. Body weights in the high-dose animals were decreased <10% in animals in the one-year interim s acrifice group, but were decreased ≥ 10% in high-dose animals in the two-year carcinogenicit y group at all determinations. Body weight gains in the high-dose animals were decreased by 11-26%, compare to th e control. Food consumption was generally decreased in the two-year carcinogenicity group at the high-dose, with sporadic decreases in the
medium-dose animals.

In a chronic toxicity/carcinogeni city study in mice, there was no treatment-related effect on mortality and no clinical signs of toxicity. Body weights were decreased in the medium- and high-dose males (500 and 1000 mg/kg/ day) by Day 147, with decreases ≥ 10% in the high-dose group. Overall body weight gains were also d ecreased by 16 and 27% in the medium- and highdose males, with no effects on body weight or body weight gain in the low-dose males (250 mg/kg/day). Consistent, dose- dependent decreases in body wei ghts were also observed in females in all treatment groups, with the decreases being ≥ 10% at selected intervals in each group. Overall body weight gains were also d ecreased by 10, 25, and 38% in the low-, mediumand high-dose females during the study.

In a chronic toxicity study in dogs, there wa s no treatment-related mortality. Dose-related emesis was noted in all treated animals, with the high-dose group exhibiting the most severity (emesis was generally similar to control at the tw o lower doses). Body weight gains in the highdose females were decreased at all time-point s through study termination. Body weight gain
decreases observed in low- and medium-dose fe males at Days 92 and 176 were not present at study termination.

In a published, non-Guideline developmental study in rats, (Kaneda et al ., 1978), ataxia was observed after dosing in dams administered o-PP at 300, 600, a nd 1200 mg/kg/day. The duration of ataxia was reported as lasting several hours, with severity incr easing in a dose-related manner. Ten of eleven dams died after 3-9 days of treatment in the high-dose group. No clinical signs of toxicity were noted at the low dose (150 mg /kg/day). Body weight gains on GDs 9, 12, 15O-PHENYLPHENOL FINAL

Page 24 of 56 and 20 were decreased in the medium-low and medium-high dose groups. Body weight gain also was decreased in the single surviv ing animal in the high-dose group.

In a non-guideline freshwater fish reproduc tion toxicity study with fathead minnows ( Pimephales promelas ), no mortalities occurred during the exposure period. There were no reported clinical signs of toxicity or significant e ffects on length or weight.

B. Estrogen Pathway

The Tier 1 in vitro ER binding assay was equivocal and positive for ER ac tivation (Routledge and Sumpter, 1997 and Miller et al. , 2001). Additionally, o-PP inhibited aromatase activity and increased estradiol production in the Tier 1 ar omatase and steroidogenesi s assays, respectively. However, in the mammalian in vivo assays, there was no convinci ng evidence for an interaction with the estrogen pathway. The uterotrophic a ssay was negative, and in the female pubertal assay the sole effect observed was a decrease in the percent regularly cycling animals at the high dose compared to the controls which occurred in the presence of systemic toxicity. Due to the lack of redundancy of responses across the mammalian assays, ther e is insufficient evidence for a potential interaction in vivo in mammals.

However, there are indications of potential effect s on the reproductive status in fish that included decreases in female vitellogeni n, fecundity and fertility in the FSTRA; there were no effects on GSI or gonadal histopathology. In another fish reproduction study with the same species as the FSTRA, decreases in egg produc tion were observed. These in vivo effects, along with indications of potential in vitro effects including ER activatio n and altered steroidogenesis, suggest that o-PP may potenti ally interact with the es trogen pathway in fish.

C. Androgen Pathway

There was evidence of potential interac tion with the androgen pathway in the in vitro AR binding assay and conflicting ev idence in the steroidogenesis a ssay where a 30% increase in testosterone production at 10 μM o-PP and a 30% decrease at 100 μM o-PP. Studies published in the open literature showed o-PP was not an androgen agonist in the MDA-kb2 cell assay, however an anti-androgen effect of o-PP wa s observed with this cell line.

The Hershberger assay was negative, and in the male pubertal assay, decreases seen in a number of androgen-dependent tissues we re observed in the presence of overt toxicity. In the FSTRA, decreases in fertility were observed, however, no other androgen-related e ffects were observed in this study or the OSRI fish reproduction study. Th ere were no androgen-related effects observed
in the Part 158 mammalian studi es. Therefore, there is no convincing evidence for potential interaction with the androgen pathway.O-PHENYLPHENOL FINAL

Page 25 of 56 D. Thyroid Pathway

There was no evidence of potential interaction of o-PP with the thyroid pathway. In female pubertal assay, there were no treatment-related effects on thyroid hormones (T4 and TSH), thyroid weights or thyroid histopathology. In th e male pubertal assays the only thyroid-related
effect observed was a decrease in serum T4 levels at all doses. In the AMA, no treatment-related finding were observed except for a significant reduction in Day 21 normalized hindlimb length; however, given the lack of eff ects on developmental stage or thyroid histopathology, this finding
alone is not considered to be sufficient evidence of a potential interaction of the thyroid pathway in amphibians. No thyroid effects were noted in any of the Part 158 mammalian studies.

E. Conclusions ! The conclusion of the WoE evaluation is that o-PP demonstrates no convincing evidence of potential interaction with the estr ogen, androgen or thyroid pathways in vivo in mammals. There is also insufficient evidence of a potential intera ction of the thyroid pa thway in amphibians. However, there is evidence of potential interaction with the estrogen pathway in non-mammalian species as suggested by the decreases in reprodu ctive output in the FSTRA and fish OSRI. These findings in the fish assays along with the results in the in vitro assays, suggest potential interaction with the estrogen path way in fish. There is no convi ncing evidence of a potential interaction with the androgen pathway in fish.

V. EDSP Tier 2 Testing Recommendations

Based on weight of evidence considerations , mammalian EDSP Tier 2 testing is not
recommended for o-PP since there was no convinci ng evidence of potential interaction with the estrogen, androgen or thyroid pathways.

Effects on fecundity/fertility were observed at 876 μg a.i./L in the FSTRA with effects on reproduction reported at 36 and 293 μg a.i./L in th e fish OSRI assay (which was similar to the FSTRA in test design). Current ly, there are no acceptable 40 CFR Part 158 chronic toxicity data for fish. At this time, based on the reproductive responses observed in the fish assays and the in

vitro assays, EDSP Tier 2 Medaka Extended One-Generation Repr oduction Test (MEOGRT) is recommended to better understand potenti al reproductive e ffects in fish.

For birds, Part 158 avian reproduction studies w ith both northern bobwhite quail and mallard duck are not available for o-PP; however, were requested under the final workplan for the registration review for o-PP (USEPA 2014). The type of data obtained from Part 158 avian reproduction studies (OCSPP 850.2300) are consid ered sufficient for evaluating potential reproductive effects to birds from o-PP exposure. Relative to the EDSP, additional testing is not
recommended.O-PHENYLPHENOL FINAL

Page 26 of 56 VI. References

Blair, R.M., Fang, H., Branham, W.S., Hass, B. S., Dial, S.L., Moland, C.L., Tong, W., Shi, L., Perkins, R., and Sheehan, D.M. (2000) The estrog en receptor relative binding affinities of 188
natural and xenochemicals: stru ctural diversity of ligands. Toxicological Sciences 54:138-153.

Caunter, J.E. and Williams, T.D. (2002) Prevento l 0 extra: determination of effects on the reproduction of fathead minnow ( Pimephales promelas ). Unpublished study conducted by Brixham Environmental Laboratory, AstraZ eneca UK Limited, UK. Study number: AH0603A. Study Sponsor: Bayer AG, Germany. (MRID 48135404)

Coady, K.K. and Sosinski, L.K. (2012) Eval uation of Ortho-Phenylphenol in the Human Recombinant Aromatase Assay. Unpublished study conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project Study ID No.: 111007, January 3, 2012. (MRID 48672608)

Cosse, P.F., Stebbins, K.E., Stott, W.T., Johnson, K.A, and Atkin, L. (1990) OrthoPhenylphenol: Palatability/probe, four week and one-year oral toxicity studies in beagle dogs. Unpublished study conducted by DOW Chem . Co., Study ID: K-001024-039, September 24, 1990. (MRID 41656401)

Eigenberg, D.A. (1990) Two-Generation di etary reproduction study in rats using orthophenylphenol. Unpublished study conducte d by Mobay Corp., September 17, 1990. (MRID 41729401; also submitted under MRID 40976501)

Eigenberg, D.A. and Lake, S.G. (1995) Two-ge neration dietary reproduction study in SpragueDawley rats using technical grade ortho-phenylphenol. Unpub lished study conducted by Bayer Corporation, Agriculture Divi sion, Toxicology, Stilwell, KS. Laboratory Study No.: 93-672- VX, September 28, 1995. (MRID 43928801)

John, J.A., Murray, F.J., Rao, K.S., and Schwetz, B.A. (1981) Teratological evaluation of Orthophenylphenol in rats. Fundamental and Applied Toxicology 1, 282-286. (MRID 00067616 and 00164362)

John, J.S., Murray, F.J., Crawford, A.A., Murray, J. S., Pilny, M.K., Schlachter, M.M., and Rao, K.S. (1990) Phase 3 Reformat of MRID 00066716/00164362: The effect(s) of orally administered orthophenylphenol on rat embr yonal and fetal development: Project HET K0001024-33(R). (MRID 92154037)

Kaneda, M., Teramoto, S., Shingu, A, Shirasu, Y. (1978) Teratogenicity and Dominant-Lethal studies with o-Phenylphenol. J. Pesticide Sci . 3:365-370.O-PHENYLPHENOL FINAL

Page 27 of 56 LeBaron, M.J., Kan, H.L., Perala, A.W. (2012) Ev aluation of Ortho-Phenyl phenol (o-PP) in the In Vitro Steroidogenesis Assay. Unpublished st udy conducted by Toxicology & Environmental Research and Consulting, Midland, MI. La boratory Report No.: 111014, Jan 4, 2012. (MRID 48672601)

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2011) Evaluation of orthophenylphenol (oPP) in an in vitro androgen receptor bindi ng assay. Unpublished study conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111114, December 19, 2011. (MRID 48672603)

LeBaron, M.J., Schisler, M.R., and Visconti, N.R. (2012) Evaluation of orthophenylphenol (oPP) in an in vitro estrogen receptor binding assay. Unpublished study conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111120, January 4, 2012. (MRID 48672604)

Lehman C.M., Fiting, J.A., Hutchinson, K.L., L ouch, D.W., Malowinski, N.A., McFadden, L.G. and Thomas, J. (2012) Ortho-Phenylphenol: The Fi sh Short-term Reproduction Assay Using the Fathead Minnow, Pimephales promelas . Unpublished study conducted by Toxicology and Environmental Research and Consulting, Midland, Michigan. Lab Study No.: 111032. Study sponsored by o-PP EDSP Consortium, Dow Chemical Company, Midland, Michigan and LANXESS Corporation, Pittsburgh, Pennsylvani a. Study completed January 04, 2012. (MRID 48672606)

Marty, M.S., Andrus, A.K., and Sura, R. (2012) Ortho-Phenylphenol: Pubertal Development and Thyroid Function in Intact J uvenile/Peripubertal Male Crl: CD(SD) Rats. Unpublished study conducted by Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laborat ory Project Study ID No.: 111088; April 13, 2012. (MRID 48686602)

Marty, M. S., Marshall, V. A., and Jeong, Y. C. (2012) Ortho-phenylphe nol: Hershberger assay in castrated male Crl:CD(SD) rats. U npublished study conducted by Toxicology & Environmental Research and Consulting, Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111084, January 5, 2012. (MRID 48672602)

Marty, M. S., Zablotny, C. L., Passage, J. K ., and Jeong, Y.C. (2012) Ortho-phenylphenol (oPP): uterotrophic assay in the immature female Crl:CD(SD) rat. Unpublished study conducted by Toxicology & Environmental Research and C onsulting, Dow Chemical Company, Midland, MI. Laboratory Project Study ID: 111086, January 5, 2012. (MRID 48672605)

Marty, M.S., Zablotny, C.L., and Sura, R. (2012) Ortho-Phenyl phenol: Pubertal Development and Thyroid Function in Intact Juvenile/Peri pubertal Female Crl: CD(SD) Rats. Unpublished study conducted by Toxicology and Environm ental Research and Consulting, The DowO-PHENYLPHENOL FINAL

Page 28 of 56 Chemical Company, Midland, MI. Laboratory Project Study ID No.: 111087; April 13, 2012. (MRID 48686601)

Miller, D., Wheals, B., Beresford, N., and Sumpte r, J. (2001) Estrogeni c activity of phenolic additives determined by an in vitro yeast bioassay. Environmental Health Perspectives 109:

133-138.

Orton, F., Rosivatz, E., Scholze, M., and Kortenka mp, A. (2011) Widely used pesticides with previously unknown endocrine activity revealed as in vitro antiandrogens. Environ Health Perspect . 119(6):794-800.

Quast, J.P. and McGuirk, R.J. (1995) Or tho-Phenylphenol: Two-year dietary chronic toxicity/oncogenicity study in B6C3Fl mice. Unpublished study conducted by DOW Chem. Co., Study ID:K-001024-047, February 1, 1995. (MRID 43545501)

Rehmann, K., Schramm, K-W., and Kettrup, A.A. (1999) Applicability of a yeast oestrogen screen for the detection of oestrogen-li ke activities in environmental samples. Chemosphere 38(14): 3303-3312.

Routledge, E. and Sumpter, J. ( 1997) Structural features of al kylphenolic chemicals associated with estrogenic activity. The Journal of Biological Chemistry 272(6): 3280-3288.

Schisler, M.R. (2012) Data Evaluation Record (DER) for: Evaluation of orthophenylphenol (oPP) in an in vitro androgen receptor bindi ng assay. Unpublished study conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111114, December 19, 2011. (MRID 48618303)

Schisler, M.R. (2012) Data Evaluation Record (DER) for: Evaluation of orthophenylphenol (oPP) in an in vitro estrogen receptor binding assay. Unpublished study conducted by Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Laboratory Project ID: 111120, January 4, 2012. (MRID 48618304)

USEPA (2010) O-Phenylphenol - Re port of the Endocrine Disruptor Review Team; Test Order# EDSP-064103-225 and 226. December 20, 2010. Office of Chemical Safety and Pollution
Prevention. U.S. Environmental Pr otection Agency, Washington, D.C.

USEPA (2011) Endocrine Disrupt or Screening Program – Wei ght of Evidence: Evaluating Results of EDSP Tier 1 Screening to Identify th e Need for Tier 2 Testing. Office of Chemical Safety and Pollution Prevention. U.S. Envir onmental Protection Agency, Washington, DC.

USEPA (2013) Tran s mittal of Meeting Minutes of the FIFRA Scientific Advisory Panel Meeting
held July 30 - August 1, 2013 on Weight-of

- Evidence: Evaluati ng Results of EDS P Tier 1O-PHENYLPHENOL FINAL

Page 29 of 56 Screening. Office of Chemical Safety and Pollution Pr evention. U.S. Environmental Protection Agency, Washington, DC.

USEPA (2014) ortho-Phenyl Phe nol (oPP) and Salts Final Work Plan. Registration Review
Initial Docket Case Number 2575. March 2014. Office of Chemical Safety and Pollution
Prevention. U.S. Environmental Protecti on Agency, Washington, DC. EPA-HQ-OPP-2013-

0524-0005.

Wahle, B.S. and W.R. Christenson (1996) T echnical Grade ortho-Phenylphenol: A Combined Chronic Toxicity/ Oncogenicity Study in the Rat. Unpublished st udy conducted by Bayer Corporation, Stillwell, KS. Study ID 92-272-SC. (MRID 43954301)

Wahle, B.S. and W.R. Christenson (1996) Suppl emental Submission to Bayer Toxicology Report No. 7908 (EPA MRID 43954301). Unpublished study conducted by Bayer Corporation, Stillwell, KS. Study ID 92-272-SC. (MRID 44832201)

Wahle, B.S. and W.R. Christenson (1996) Suppl emental Submission to Bayer Toxicology Report No. 7908 (EPA MRID 43954301). Unpublished study conducted by Bayer Corporation, Stillwell, KS. Study ID 92-272-SC. (MRID 44852701)

Zablotny, C.L., Breslin, W.J., and Kociba, R.J. (1991) Ortho-Phenylphenol (o-PP): Gavage teratology study in New Zealand White Rabbi ts. Unpublished study conducted by DOW Chem. Co. Study ID: K-001024-045, April 23, 1991. (MRID 41925003)O-PHENYLPHENOL FINAL

Page 30 of 56 APPENDIX 1. EDSP Tier 1 Screening Assays

Amphibian Metamorphosis Assay (AMA): (OCSPP 890.1100)

The 21-day assay (MRID 48916901) of ortho- phenylphenol (99.9% purity) on amphibian metamorphosis of the African clawed frog ( Xenopus laevis ) was studied under flow-through conditions. Amphibian larvae at developmental Nieuwkoop-Faber Stage (NF) 51 (80/control and treatment group; 20/replicate) were exposed to no minal concentrations of 0 (negative control), 0.024, 0.12, 0.60, and 3.0 mg a.i./L. The time-weighted average (TWA), measured concentrations were <0.00514 (<LOQ; negative control), 0.0175, 0.0673, 0.352, and 1.92 mg a.i./L. The test system was maintained at 21.3-22.8°C and a pH of 7.0-7.6.

A single mortality was observed in the high treatme nt level during the study. Over the course of the exposure, a single tadpole in the mid-low treatment level was obs erved resting on the surface, and a single tadpole in the high treatment level was observed rest ing on the bottom. There were no other signs of clinical toxicity.

Ortho-phenylphenol did not signi ficantly affect (p>0.05) Day 7 normalized (for snout-vent length) hind-limb length (HLL) at any treatmen t level; however, Day 21 HLL of non-late stage tadpoles (NF<61) was significan tly reduced (Jonckheere, p = 0.006) by 11% at the high treatment level relative to the negative contro l. Day 21 normalized HLL was also significantly reduced (Jonckheere, p = 0.036) by 8% at the high treatmen t level relative to the negative control. Among late-stage tadpol es (NF>61), there were no significant effects on normalized HLL or growth endpoints compared to the negati ve control. There was no significant (p>0.05) acceleration or delay of median NF developmental stage at Day 7 or Day 21 in any treatment group relative to the negative control. No asynchronous development was observed throughout the test. Mild follicular cell hypertrophy was obser ved in the negative control and all treatment groups, though there was no apparent treatment-related effect.

Androgen Receptor (AR) Bindi ng Assay; (OCSPP 890.1150)

In an androgen receptor (AR) binding assay (MRID 48672603), ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a competitive binding experiment, which measured the binding of a single concentration of [ 3 H]-R1881 (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 10 to 10

− 3 M) of o -phenylphenol (o-PP, 99.9% purity, Lot #CHHYDD0051). Et hanol was used as a vehicle at a final assay concentration
of <3%. A total of three runs were performed, and each run included dexamethasone as a weak positive control, and R1881 as the ligand reference standard.O-PHENYLPHENOL FINAL

Page 31 of 56 Prior to conducting the AR competitive binding e xperiments, a saturation binding experiment was conducted to determine if the AR in the cy tosol preparation was present in reasonable numbers and functioning with a ppropriate affinity for the radi olabeled ligand. Data from a saturation binding experiment were not provided in the study report; however the results from a single run of the saturation binding experiment were incl uded in a study profile (MRID 48618303) submitted by the test order recipient. In the saturation binding experiment, the maximum binding capacity (B max ) was 3.245 fmol/100 μg protein a nd the dissociation constant (K d ) was 0.4641 nM. Although these values were sli ghtly below the range of values from the validation studies, the results were highly reproducible and all ot her performance criteria and the responses in the competitive binding assays indica ted acceptable performance of the assay. The Scatchard plot indicated a linear response across th e concentrations of ligand added. Nonspecific binding as a percent of total bi nding was less than 20% across the entire concentration range in the saturation binding assa ys (range 6.2-19.8%, with the excep tion of the high concentration (10 nM) in one assay, which was 24.6%).

In the competitive binding experiment, o-PP reduced the mean specific binding of the radiolabeled ligand in a con centration-dependent manner in all three runs. The maximum binding reduction occurred at the highest concentration tested (10 − 3 M), with specific binding being reduced to 6.3-7.4%. Specifi c binding was also reduced at 10 − 4 M (62.8-68.8%), but not at
concentrations ≤ 10 − 5 M (92.8-103.7% binding). Specific [ 3 H]-R1881 binding was reduced to <50% at the highest concentrations tested in all three runs.

The estimated mean log IC 50 was

− 3.749 M for o-PP compared to that of R1881 (

− 8.970 M) and the weak positive control of

− 4.418 M. The mean RBA was 0.0006% for o-PP compared to 0.0028% for the positive control. Confidence in thes e numbers is high due to the small variation. No precipitation of the test compound was reporte d. The solvent control responses indicated no drift in the study assay. All pe rformance criteria were met.

Based on the results from the three runs, o-PP is classified as a Binder in the Androgen Receptor Binding Assay.

Aromatase Assay; (OCSPP 890.1200)

In an in vitro aromatase (CYP 19) assay (MRID 48672608), o -phenylphenol (o-PP, 99.9% a.i., lot# CHHYDD0051) was incubated with human recombinant aromatase and tritiated androstenedione ([1 β

3 H(N)]-androst-4-ene-3,17-dione; [ 3 H]ASDN) in 1% ethanol for 15 minutes to assess the potential of o-PP to inhi bit aromatase activity. o-PP was tested at logarithmic concentrations from 10 –10 M to 10

− 3 M in 3 independent runs.O-PHENYLPHENOL FINAL

Page 32 of 56 Aromatase activity was determined by measuring the amount of tritiated water produced at the
end of the 15-minute incubation for each concentration of chemical. Tritiated water was quantified using liquid scintillation counting (LSC ). Three runs were conducted and each run included a full activity c ontrol, a background activity control, a positive control series (10 –10 to
10 –5 ) with a known inhibitor (4- hydroxyandrostenedione; 4-OH ASDN), and the test chemical series (10 –10 to 10 –3 ) with three repetitions per concentration.

Aromatase activity in the fu ll activity controls ranged from 0.080 to 0.159 nmol·mgprotein

− 1 ·min

− 1 for the three test runs, with a mean and standard deviation of 0.124±0.029 nmol·mg-protein

− 1 ·min

− 1 . Activity in the background controls ranged from 2.38% to 7.88% and averaged 5.7% of the full control activity. The response of the full activity controls and background controls was acceptable.

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.108±0.054 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.002±0.004 nmol·mgprotein -1 ·min

− 1 at the highest tested concentration (10

− 5 M). These results were generally within the recommended ranges for the top of the curv e, bottom of the curv e, Hill slope, log IC 50 , and coefficient of variation for replicates of each c oncentration, with the exceptions that the overall %CV for the highest three concentrations (51.2, 36.2, and 129%) exceeded the recommended limit of 15%. The estimated log IC 50 for 4-OH ASDN averaged

− 7.1 M and the Hill slope was

− 1.17.

For o-PP, aromatase activity averaged 0.124±0.031 nmol·mg-protein

− 1 ·min

− 1 at the lowest tested concentration (10

− 10 M) and 0.007±0.013 nmol·mg-protein

− 1 ·min

− 1 at the highest successfully tested concentration (10

− 3 M). The overall % CV for the 10

− 3 and 10

− 8 M concentrations (69% and 22%, respectively) exceeded the recommended limit of 15%. The data for o-PP were modeled for all runs, and the goodness of fit (r 2 ) values were 0.87-0.98. The best fit Hill slope and log IC 50 values for o-PP across the three independe nt runs did not diffe r significantly.

The lowest portion of the response curve wa s less than 50% activity at highest two concentrations tested. Aromatase activity was 53% at 10 –4 M, and decreased to 6% at 10

− 3 M oPP. The average log IC 50 and Hill slope for o-PP were

− 3.97 M and

− 0.99, respectively.

Based on the data from the average response curv e, o-PP is classified as an Inhibitor of aromatase activity in this assay.

Estrogen Receptor (ER) Bindi ng Assay; (OCSPP 890.1250)

In an estrogen receptor (ER) binding assay (MRID 48672604) for o-phenylphenol (o-PP), uterine cytosol from Sprague Dawley rats was used as the source of ER for a competitive bindingO-PHENYLPHENOL FINAL

Page 33 of 56 experiment which measured the binding of a single concentration of [ 3 H]-17 β -estradiol (1 nM) in the presence of increasing concentrations (logarithmic increase from 10

− 10 to 10

− 3 M) of o-PP (99.9% purity, Lot # CHHYDD0051). Ethanol was used as the solvent vehicle at a final
concentration of <3%. A total of three r uns were performed, and each run included 19- norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and 17- β

estradiol as the natural ligand reference material.

Saturation binding experiments were conducted first to demonstrate that th e ER in the uterine cytosol was present in reasonable numbers and were functioning with the appropriate affinity for the radioligand. No data from the saturation binding experiment were provided in the study report; however, the results from a single run of the saturation binding experiment were included in a study profile (MRID 48618304) submitted by the test order recipient. The results from the single saturation binding run show ed a maximum binding capacity (B max ) of 59.28 fmol/100 μg protein with the disso ciation constant (K d ) of 0.1032 nM. Nonspecific binding as a percent of total binding was 1.7-8.6% across the concentration range.

In the competitive binding experiment, the estimated mean log IC 50 was

− 8.943 M for 17 β

estradiol and

− 5.454 M for the weak positive control. The mean RBA was 0.032% for the weak positive control. All performance criteria were met. o-PP reduced the mean specific binding of the radiolabeled ligand in a concentration-dependent manner in all three runs (declining to <50% in Runs 1 and 2); however, the o-PP bindi ng curve exhibited steep Hill slopes (

− 2.262 to

− 4.588), indicating that the data did not fit the four-parameter, nonlinear regression model for analysis of one-site binding. The mean estimated log IC 50 (n=2) was

− 3.192 M for o-PP; an
RBA value was not determined.

Based on the results from the thr ee runs, o-PP is classified as E quivocal in the Estrogen Receptor Binding Assay.

ER α Transcriptional Activation (ERTA) Assay; (OCSPP 890.1300)

Requirement satisfied by OSRI (Rou tledge and Sumpter, 1997, and Miller et al ., 2001); see Appendix 2 for summaries.

Fish Short Term Reproduction Assay (FSTRA); (OCSPP 890.1350)

The 21-day short-term reproduction assay (MRID 48672606) of ortho-phenylphenol (o-PP) with fathead minnows ( Pimephales promelas) was conducted under flow-t hrough conditions. Adult fish (16 spawning groups; 2 males and 4 females in each group; 5.5 months old) were exposed to o-PP at nominal concentrations of 0 (neg ative control), 0.02, 0.20, a nd 2.0 mg a.i./L; timeO-PHENYLPHENOL FINAL

Page 34 of 56 weighted average (TWA), measured con centrations were <0.00495 (<LOQ), 0.0158, 0.0755, and 0.876 mg a.i./L. The test system was maintained at 24.6 to 25.5 o C and a pH of 7.2 to 7.4.

Overall survival was significan tly decreased (p<0.05) by 29% at the high treatment level (75%
survival in males and 69% survival in female s) compared to the negative control with 100% survival in the negative contro l and all other treatment groups. Si x of seven deaths occurred in a single tank over two days, potenti ally indicating a tank-related issue (the high treatment level was well below any acute toxicity level). o-PP e xposure was not associated with increases or decreases in body weight and/or length in either sex at any treatment level (p>0.05). With the exception of a single lethargic male in the high treatment group, no effects on secondary sex characteristics or clinical signs were observed for any treatment group compared to the negative
control.

In the negative control, spawning did not occur at least every 4 days in three of the four replicates. Mean fecundity wa s 13.9 eggs/female/day/replicat e (range: 7.3-20.1); fertilization success in the negative contro l group averaged 97.2% (range: 92.4-99.7%). Two of the four negative control replicates were below the minimum fecundity criterion of 15 eggs/female/day/replicate at 7.3 and 9.2 eggs/female/reproductive day. o-PP did not significantly affect (p>0.05) fecundity or fert ility compared to the negative cont rol. However, the variances in the high treatment group for fecundity a nd fertilization success were high (7.3±6.38 eggs/female/day/replicate and 66.0±57.13%, respectively); while neit her value was significantly different (p>0.05) from the ne gative control, both were ma rkedly decreased by 47.5 and 32%, respectively, compared to the negative control, and these differences were considered biologically significant.

Female vitellogenin (VTG) was significantly redu ced (p<0.05) by 35.5% rela tive to the negative control at the high treatment le vel; male VTG was unaffected by o-PP treatment. There were no other significant effects on endpoints ( e.g. , nuptial tubercle score, gonado-somatic index (GSI), or gonadal staging). There were no treatment-relate d histopathological observations. In females, there was an increase in the incidence and severity of oocytes atresia in tr eated fish compared to the negative control; howev er, the effects lacked a concentra tion-response relationship. Plasma
testosterone and 17 β -estradiol levels we re not measured.

Hershberger Assay; (OCSPP 890.1400)

In a Hershberger Assay (MRID 48672602) sc reening for androgenic activity, orthophenylphenol (o-PP; 99.9% a.i., Lot # CHHYDD0051) in corn oil was administered daily via oral gavage (4 mL/kg) to groups of seven 55-da y old, castrated male Sp rague Dawley rats at dose levels of 0 (vehicle), 50, 250, or 1,000 (lim it dose) mg/kg/day. An androgenic positiveO-PHENYLPHENOL FINAL

Page 35 of 56 control group consisted of seve n castrated rats exposed to 0. 4 mg/kg/day of testosterone propionate (TP) by subcutan eous (s.c.) injection.

To screen for potential anti-a ndrogenic activity, o-PP (99.9% a.i ., batch/lot # CHHYDD0051) in corn oil was administered daily via oral gava ge (4 mL/kg) to groups of seven 55-day old, castrated male Sprague Dawley rats at dose le vels of 0 (vehicle), 50, 250, or 1,000 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by s.c. injection. The anti-androgenic positive control group consisted of seven castr ated rats exposed to 0.4 mg/kg/day TP and 3 mg/kg/day fl utamide (FT). TP alone was used as the anti-androgenic negative control.

For both components of the assa y, body weights and food consump tion were determined daily. The animals were dosed for 10 consecutive days a nd terminated approximately 24 hours after the final dose administration. At necropsy, the fi ve androgen-dependent ti ssues, adrenal glands, kidneys, and liver were co llected and weighed.

All animals survived until sc heduled termination. One anim al in the high dose group was observed to have mottled lungs at necropsy, indicati ng a gavage error; therefore, the results for this animal were excluded from the study. Th ere were no treatment-related clinical signs in
animals dosed at ≤ 250 mg/kg/day in either assay. In bot h assays, the majority of high-dose animals (6/7 androgen assay; 5/7 in anti-andr ogen assay) had some or all of the following observations: clear or red peri oral soiling, perineal soiling (u rine and/or feces), decreased activity, noisy respiration, and soft feces. At necropsy, one animal in each high dose group (both assays) was found to have thickening of the nonglandular mucosa of the stomach.

In the androgen agonist assa y, body weights were decreased (p <0.05) by 15% on Day 7 in the 1,000 mg/kg/day group compared to controls; however, body wei ghts at the end of the study were similar to controls. Food consumption in this group was decreased (p<0.05) by 42% during Days 4-7. Body weights and body weight gain s in the 50 and 250 mg/kg/day groups were comparable to controls throughout the duration of the study; food consumption was increased (p<0.05) in the 50 mg/kg/day group on Days 1-4 ( ↑ 6%). In the positive control group, cumulative body weight gains were increased (p<0.05) on Days 1-4 ( ↑ 45%), Days 1-7 ( ↑ 50%), and Days 1-11 ( ↑ 36%); food consumption was increased (p<0.05) in this group on Days 1-4 ( ↑ 14%), Days 4-7 ( ↑ 14%), and Days 7-11 ( ↑ 12%). There were no trea tment-related effects onweights of the accessory sex organs/tissues, adrenal glands, kidneys, or liver for any o-PPtreatment group. Animals in the TP group had increased (p<0.05) accessory sex organ weightsas follows: 1760% in seminal vesicles; 1117% in ventral prostate; 226% in levator anibulbocavernosus (LABC); 806% in Cowper’s gland; and 67% in glans penis. Adrenal glands, kidney, and liver weights in the positive control group were comparable to the vehicle control.O-PHENYLPHENOL FINAL

Page 36 of 56 In the anti-androgen assay, body weights and body weight gains in the o-PP treatment groups and positive control group were comparable to the negative control ov er the duration of the study. In the 1,000 mg/kg/day group, food consum ption was decreased (p<0.05) by 19% during Days 4-7. Food consumption in the 50 and 250 mg/kg/day groups and the positive control group
was similar to controls over the duration of the study. At 1,000 mg/kg/day, the weights for the ventral prostate were decreased (p<0.05) by 28% compared to the controls. However, there were
no treatment related effects on th e weights of the seminal vesicles, LABC, Cowper’s glands, glans penis, adrenal glands, ki dneys, or liver for any o-PP-treat ment group. Animals dosed with TP + FT (positive control) had decreased (p<0.05) accessory sex organ weights as follows: 83% in seminal vesicles; 77% in ventral prostate; 46 % in LABC; 61% in Cowpe r’s gland; and 18% in glans penis. Adrenal glands, kidney, and liv er weights in the positive control group were comparable to the vehicle control.

Percent CVs for the vehicle control and the 1,000 mg/kg/day treatment groups for both assays were compared to the performance criteria in the guidelines. A ll %CV values were less than the maximum recommended values, with the exception of the %CV for Cowper’s glands for the control group in the androgen agonist assa y (CV = 88%; maximum recommended CV = 55%) and the %CV for LABC for the high dose group in the anti-androgen assay (CV = 22%; maximum recommended CV = 20%).

Statistically significant changes were not seen in two or more of the five androgen sensitive tissue weights. o-PP was negative for androgeni city and anti-androgenicity in the Hershberger

assay.

Female Pubertal Assay; (OCSPP 890.1450)

In a Female Pubertal Assay (MRID 48686601), 15 Sprague Dawley Crl:CD(SD) rats/dose group were treated daily via oral gavage with O -phenylphenol (o-PP; 99.9% purity, Lot No. CHHYDD0051) in corn oil at doses of 0 (vehicle), 50, 250, or 900 mg/kg/day from post-natal day (PND) 22 to 42. Animals were examined fo r vaginal opening (VO) daily beginning on PND 22, and the body weight at day of attainment was recorded. Following sacrifice on PND 42, thyroxine (T 4 ) and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay or chemiluminescent immunoassa y. Liver, pituitary, adrenals, and urogenital organ weights were recorded, and microscopic examinations were performed on the ovaries, uterus, thyroid, and kidneys.

All animals survived until scheduled termination, with the exception of one animal in the highdose group (death due to gavage error). Addition ally, no treatment-relate d clinical signs of toxicity were observed in animals for any dose groups.O-PHENYLPHENOL FINAL

Page 37 of 56 Mean body weights were similar across all dose groups on PND 21. There were no effects on age at attainment of VO for any dose of o-PP. Initial a nd terminal body weights, body weight gains, and body weight at attain ment of VO were similar across all dose groups. However, the 900 mg/kg/day group weighed 8% less than cont rol animals at PND 35. The 900 mg/kg/day group gained 10% to 13% less th an control between PND 22-35, but these decreases were not statistically (NS) different from control animals. High-dose rats ga ined more weight than control animals during the PND 35-42 interval, such that body weight gains were onl y slightly decreased (3.2%, NS) over the entire dosing period (PND 22-42).

At 900 mg/kg/day, modest decreases (p<0.05) were noted in adjusted pituitary ( ↓ 12%) and adrenal ( ↓ 13%) weights, and a minor incr ease (p<0.05) of 9% was noted in relative liver weight. Relative pituitary weights were decreased (p<0.0 5) by 10%. There were no dose-related effects on the weights of kidneys, ova ries, uterus, or thyroid.

There were no effects of treatment on mean age at first estrus or mean cycle length. In the highdose animals, the percent cycling was decreased (NS) relative to the c ontrol from 93.3% to 64.3%, and the percent regularly cycling was de creased (p<0.05) from 86.7% to 28.6%. However, the estrous cycle status at necropsy (d iestrus, proestrus, es trus, not cycling) was generally similar across all groups.

Serum urea nitrogen (BUN) and phosphorus leve ls were increased (p <0.05) in the 900 mg/kg/day dose group ( ↑ 23.1 and ↑ 14.1%, respectively). These increases were considered treatment-related, and were associated with tr eatment-related kidney effects. Significantly increased alanine aminotransferase activity (ALT ; p<0.05; 102%) was also considered treatmentrelated.

o-PP was not associated with any significant treatment-related ch anges in thyroid histopathology. At 900 mg/kg/day, increased incide nces of the following histologica l lesions were noted in the kidney: (i) multifocal renal tubular dilatation (1 2/14 treated animals, very slight and slight severity vs. 4/15 controls, very slight sever ity); (ii) multifocal collecting duct epithelium hypertrophy (3/14 treated animals, very slight severity vs. 0/15 controls); (iii) multifocal renal tubule dilatation (9/14 treated animals, very slight to slight severity vs. 1/15 cont rols, very slight severity; and (iv) slight focal renal tubular necrosis, with accompanying inflammation (2/14
treated animals vs. 0/15 controls).

The doses tested were adequa te based on the increased serum ALT, BUN, phosphorus, renal lesions, and effects on estr ous cyclicity seen at th e high dose (900 mg/kg/day).O-PHENYLPHENOL FINAL

Page 38 of 56 Male Pubertal A ssay; (OCSPP 890.1500)

In a Male Pubertal Assay (MRID 48686602), 16 Sprague Dawley Crl:CD(SD) rats/dose group were treated daily via oral gavage with O -phenylphenol (o-PP; 99.9% purity, Lot No. CHHYDD0051) in corn oil at doses of 0 (vehicle), 50, 250, or 900 mg/kg/day from post-natal day (PND) 23 to 53. Animals were examined fo r preputial separation (PPS) daily beginning on PND 30, and the body weight at day of attainme nt was recorded. Following sacrifice on PND 53, total serum testosterone, thyroxine (T 4 ), and thyroid stimulating hormone (TSH) levels were analyzed by radioimmunoassay or chemiluminescen t immunoassay. Liver, pituitary, adrenal and urogenital organ weights were recorded, and mi croscopic examinations were performed on the testes, epididymides, thyroid, and kidneys.

All animals survived until schedul ed termination, with the excepti on of four animals. The cause of death for three of these animals (one in each o-PP-dose group) was considered to be gavage
error. The cause of death of the fourth anim al (high-dose group) was not determined, but was not attributed to o-PP treatment. Additionally, no treatment-related clinical signs of toxicity were observed in animals for any dose groups. Ad justed terminal body weights were decreased by 8% in the 900 mg/kg/day group, due to a trea tment-related effect on body weight gain ( ↓ 13%; p<0.05) over the duration of the st udy. Body weights and body weight gains were not affected at

≤ 250 mg/kg/day. The adjusted age of attain ment of PPS was delayed by 1.6 days (not significant; NS) in the 900 mg/ kg/day group compared to the c ontrol (45.1 vs. 43.5 days). Delayed PPS appeared to be related to the decr eased terminal body weight and body weight gain observed in the high-dose group.

Significant (p<0.05) organ weight changes after administratio n of o-PP included increased relative liver weight at 250 mg/kg/day ( ↑ 8%), and adjusted and re lative liver weights at 900 mg/kg/day ( ↑ 10% and ↑ 21%, respectively). Compared to the control, unadjusted and adjusted pituitary weights were decreased by 15% and 10%, respectively, in the 900 mg/kg/day group, and unadjusted adrenal wei ghts were decreased by 11% at ≤ 250 mg/kg/day and by 16% at 900 mg/kg/day. However, the most relevant measur es are the adjusted weights, and only minor increases (p<0.05) were noted in the liver and adrenal at 900 mg/kg/day.

Additionally, decreased (p<0.05) organ weights at 900 mg/kg/day included: unadjusted weights of seminal vesicles + coagulating glands with and without fluid ( ↓ 23% and ↓ 18%, respectively); unadjusted and adjusted ventra l prostate and LABC weights ( ↓ 17-20% and ↓ 16-18%, respectively); unadjusted weights for the left and right epididymides ( ↓ 10% and ↓ 9%, respectively); and unadjusted and adjusted weights for the left testis ( ↓ 9% and ↓ 7%, respectively). As adjusted weight decreases we re observed in three andr ogen sensitive tissues,
these effects were considered treatment-relate d. There were no dose-related effects on the weights of kidneys, dorsolateral pros tate, right testis, or thyroid.O-PHENYLPHENOL FINAL

Page 39 of 56 No treatment-related effects were observed on serum testosterone or TSH concentrations. Serum

T 4 concentrations were decreased (p<0.05) at all dose levels by 15-23%, but without clear dosedependence. There were no gross or histological lesions of th e thyroid. At 900 mg/kg/day, an apparent treatment-related effect was observe d on serum urea nitrogen concentrations, which corroborated the renal histological evidence. Significantly incr eased alanine aminotransferase (ALT) activity ( ↑ 95%) was considered treatment-relate d, but the 32% increase in aspartate aminotransferase activity (AST) was a slight response and not adverse. The 40% decreases (p<0.05) in gamma glutamyl transferase ( GGT) activities at 250 and 900 mg/kg/day are considered slight and were not dose-dependent.

No treatment-related findings were observed in the right testes or right epididymides at any o-PP dose. However, in the kidneys, there were in creased incidences of renal tubular dilatation (combined focal and multifocal, very slight a nd slight severity) at 900 mg/kg/day (12/14 animals) compared to controls (5/15 animals) . Increased incidences of collecting duct epithelium hypertrophy (combined focal and multifocal , very slight severity) were also observed at 900 mg/kg/day (5/14) compared to controls (0 /15). Although the incide nces of renal tubular degeneration were similar in high-dose and contro l animals, the severity was increased at the high dose; the incidence of slight multifocal tubular degeneration was 3/14 at 900 mg/kg/day compared to 0/15 in controls.

The high dose of 900 mg/kg/day was sufficient based on the 8% decrease in the adjusted terminal body weight, the 13% decrease overa ll body weight gain, the 7-17% decreases in androgen-sensitive organ weights, the 94% increase in serum ALT, and increased incidences and/or severity of renal lesions.

Steroidogenesis Assay; (OCSPP 890.1550)

In a steroidogenesis assay (M RID 48672601), H295R cells cultured in vitro in 24-well plates were incubated with ortho-phenylphenol (o- PP, 99.9% a.i., Lot# CHHYDD0051) at log concentrations from 0.0001 to 100 μM in triplica te for 48 hours. Dimethyl sulfoxide (DMSO) was used as a vehicle, at a fi nal assay concentration of 0.1%.

Testosterone and estradiol levels were measur ed by LC/APPI-MS/MS. A Quality Control (QC) plate was run concurrently with ea ch independent run of a test chem ical plate to demonstrate that the assay responded properly to pos itive control agents at two con centration levels. The positive
controls included a known inhibito r (prochloraz) and a known indu cer (forskolin) of estradiol and testosterone production.O-PHENYLPHENOL FINAL

Page 40 of 56 No precipitation was observed at o-PP concentration up to 100 μ M, and o-PP was not cytotoxic to the H295R cells. Cell viability for the QC and test plates was ≥ 95.4% for o-PP and the
reference standards.

Four independent test runs of the steroidogenesis assay with o- PP were conducted, but the fourth was not statistically evaluate d as three acceptable independent assays for o-PP had been completed. The minimum basal hormone production levels in the solvent control (SC) wells were acceptable for testosterone, but basal prod uction of estradiol was below the recommended 40 pg/mL level. However, basal estradiol produc tion was still >26 pg/mL which is >2.5-fold the minimum detection limit (MDL), as per Guideline recommendation. Forskolin at 10 μ M
induced testosterone and estr adiol production as required ( ≥ 2-fold and ≥ 7.5-fold, respectively),
and 1 μ M prochloraz inhibited testosterone production by >50% as required; however, inhibition of estradiol production (40%) was below the required 50%.

The intra- and inter-plate vari ability (%CV) was reported to be 0.5-24.4% for the testosterone and estradiol in the test runs, with the exception of 0.01 μM o-PP in Run 3 where the intraplate CV was 39.9%, due to in creased responsiveness.

Conflicting results were observed for testosterone production, with a statistically significant 30% increase at 10 μM o-PP and a 30% decrease at 10 0 μM o-PP. Statistically significant increases in estradiol production were obser ved at 10 μM o-PP (2.6-fold) and 100 μM o-PP (4.0-fold). These effects on estradiol were reproducible a nd concentration-dependent in all three runs.

Based on the hormone responses in the three in dependent runs, o-PP treatment resulted in statistically significant and repr oducible increases in estradio l and testosterone production; however, the alterations in testoste rone production were inconsistent in direction of the response.

Uterotrophic Assay; (OCSPP 890.1600)

In a Uterotrophic Assay (MRID 48672605) conducted to screen for potential estrogenic activity, ortho-phenylphenol (o-PP; 99.9% a.i., Lot # CHHYDD0051) in corn oil was administered daily via oral gavage to groups of six immature fema le Sprague Dawley rats at dose levels of 0 (vehicle), 50, 250, or 1000 (limit dose) mg/kg/day on post-natal days (PND) 19-21. A positive control group was treated with a daily dose of 17 α -ethynyl estradiol (EE) at 10 μ g/kg/day by oral gavage. Body weights were determined daily. All animals were examined for precocious vaginal opening, and then terminated and necropsied on PND 22 approximately 24 hours after
the final dose to determine wet a nd blotted uterin e weights.

All animals survived until sche duled termination. No treatment -related clinical findings or precocious vaginal opening were observed in o- PP dosed animals. Overall body weight gainsO-PHENYLPHENOL FINAL

Page 41 of 56 were decreased (p<0.05) by 25% in the 1000 mg/ kg/day group. Body weights in the 50 and 250 mg/kg/day dose groups and the positive control group were comparable to controls throughout the duration of the study. Uterin e weights in the o-PP treatment groups were comparable to the
vehicle controls. Absolute wet and blotted ut erus weights for the positive control (EE) group were increased (p<0.05) by 897% and 432%, respectively, as expected.

No statistically significant changes were seen in uterine weight in this as say. o-PP is negative in the uterotrophic assay.O-PHENYLPHENOL FINAL

Page 42 of 56 APPENDIX 2. Other Scientifically Relevant Information (OSRI)

Published literature and Part 158 ge neral toxicity studies considered relevant for determining the potential interaction of o-PP with the endocri ne system are summarized below. Given the breadth of toxicity data available on o-PP, the only Part 158 studies disc ussed in this appendix are those that fulfill current guideline requirements and are directly relevant to assessing potential endocrine effects ( i.e. , multiple-generation rodent reproductive studies, development studies, chronic toxicity studies, etc.).

AR Transcriptional Activation (ARTA)

In a published study by Orton et al . (2011) , several chemicals, in cluding o-PP (>97% purity)
were tested for endocrine-d isrupting effects in an in vitro ARTA (MDA-kb2) assay. The chemicals were dissolved in ethanol to prepare stock solutions (final et hanol concentration was not reported). The MDA-kb2 assay is conducte d with human breast cancer cells stably transfected with a fire-fly luciferase reporter ge ne that is driven by androgen-response elementcontaining promoter.

The MDA-kb2 cell concentration was 1×10 5 cell/mL in phenol red- free Leibowitz-15 medium with 10% (charcoal-stripped) feta l calf serum. Cells were seed ed and allowed to attach for 24 hours, followed by exposure to eight serial diluti ons of o-PP (in range of 0.01 to 100 μM) with or without 0.25 nM dihydrotestosterone (DHT) for an additional 24 hours. On each plate, the controls included media, ethanol, DHT co-e xposure (0.25 nM), DHT serial dilutions (0.002- 10 nM), and flutamide (0.013-8 μM) or procym idone (0.005-3.2 μM) serial dilutions. All
concentrations were tested in duplicate with two plates.

In the MDA-kb2 cell assay, o-PP was negative as an androgen agonist, but an anti-androgen IC 20 of 3.43 μM was determined for o-PP. o-PP was not cytotoxic at the tested concentrations (up to 50 μM); therefore, the concentration required to yield cytotoxicity was greater than the
concentration associated w ith anti-androgenicity.

ERTA/ER Binding/Cell Proliferation

In a published study, Blair et al . (2000) o-PP was tested in an ER competitive binding assay to determine ER affinity. The source of the ER for this assay was rat uterine cytosol. Chemicals were first screened at a high concentration and those that were positive for binding were further tested by using a range of concentrations to generate a binding curve and calculate the IC 50 for
each chemical. o-PP (99% purity) was reported to be negative fo r ER binding at a concentration
of 1×10

− 4 M.

In a published study, Routledge and Sumpter (1997) evaluated 29 alkylphenolic compounds, including o-PP (99% purity) and ot her isomers of phenylphenol, for estrogenic activity with anO-PHENYLPHENOL FINAL

Page 43 of 56 in vitro yeast assay. The yeast strain employed was stably transfected to express the human ER α (hER α ) gene together with expression plasmids (containing estrogen responsive elements and the

lac -Z reporter gene encoding the enzyme β -galactosidase) . Chlorophenol red- β -Dgalactopyranoside (CPRG) was used as the chro mogenic substrate to measure receptor binding activity. o-PP, serially diluted in ethanol (final ethanol concentr ation was not reported)

, was evaluated at concentrations of 800 μM to 200 nM; solvent and 17 β -estradiol controls were included in the assay . The estrogenic activity of the diffe rent isomers of phenylphenol (4-, 3-, and 2-phenylphenol) indicated that the most active derivative, 4-phenylphenol, was 20,000 times less potent than 17 β -estradiol. The study authors reported that each successive movement of the phenyl group around the phenolic ring resulted in an approximately 7-fold reduction in activity. o-PP (2-phenylphenol) was the leas t active isomer and was estimated by the reviewers (based on 4-phenylphenol and a 14-fold reduction in act ivity) to be 280,000 times less potent than

17 β -estradiol.

In a published study, Rehmann et al. (1999) tested thirty compounds, including o-PP (purity not reported), for estrogenic activity with recombinant yeast cells expressing a fusion protein containing the hER binding domain and the GAL4 DNA binding domain to activate transcription
of the β -galactosidase reporter gene. o-PP was te sted at 10 nM, 10 μM, and 1 mM in three independent experiments and was re ported to be negative for hER ac tivation in this test system.

In a published study, Miller et al. (2001) evaluated 73 phenolic additives, including o-PP ( ≥ 98% purity), for estrogenic activity with an in vitro yeast assay. The yeast strain employed was stably transfected to express the hER α gene. Chlorophenol red- β -D-galactopyranoside (CPRG) was used as the chromogenic substrate to measure receptor binding activity. Stock solutions of the test chemicals were serially dilu ted in ethanol for the assay (fin al ethanol concentration was not reported); 17 β -estradiol and solvent contro ls were included with each assay. Each chemical was
tested at least twice to ensure consistency of the data. The ED 50 (effective dose) was calculated by using the optical density that gave a 50% resp onse relative to the reference standards. The

ED 50 for 17 β -estradiol was 2.0 × 10

− 10 M (±0.22 × 10

− 10 M; n=14). o-PP exhibited weak estrogenic activity (1 /2,000,000 relative to 17 β -estradiol) in the in vitro yeast assay.

In a published study, Soto et al. (1997) developed a method to extract and separate xenoestrogens from ovarian estrogens in hum an serum, followed by determination of the cumulative xenoestrogen load (as estradiol eq uivalents) in the E-SCREEN bioassay. The chemicals tested included o-PP (purity not reported) and its meta- and para- isomers (4-, 3-, and 2-phenylphenol) . The test substances were initially identified and ranked by estrogenic effect using the E-SCREEN bioassay, after which thos e with more potent activity were used in development of the test method a nd in further testing for cumulati ve effects using the bioassay.

In the E-SCREEN assay, cloned MCF-7 cells we re plated with the experimental medium containing estradiol (E 2 ) or a range of concentrations of th e test compounds di ssolved in ethanolO-PHENYLPHENOL FINAL

Page 44 of 56 or DMSO. The assay was terminated 6 days af ter initiation (late expone ntial phase) by removing
the medium from the wells and ad dition of a cell-lysing solution. Cell yield achieved at different concentrations of the test substances compared to the hormone free control was determined by counting cell nuclei. Estrogenic activity of th e test chemicals was assessed by determining the relative proliferative potency (R PP) and the relative proliferative effect (RPE). The RPE indicated whether or not the test compound induc ed a proliferative re sponse quantitatively
similar to the one obtained with E 2 (full agonist, RPE=100) or a proliferative yield lower than
that obtained with E 2 (partial agonist). In the initial E-SCREEN assay, o-PP was shown to have minimal estrogenic activity and was a partial agonist (RPE = 30% and RPP = 0.0001%) at a test concentration of 100 μM (the lowest con centration needed for maximal effect).

In a published study, Itoh et al. (2000) investigated the estrogenic effects of sixteen chemicals, including o-PP (purity not reported), with the MV LN assay, which utilizes an MCF-7 derivative that has been stably transfected with the Vit-Luc reporter gene. The MVLN cell line expresses the endogenous ER of MCF-7 and contains an exogenous estrogen-responsive reporter (luciferase); therefore, the estrog en-specific transcription activity of a test chemical is directly related to the activity of luciferase measured in the lysate of treated MVLN cells.

In the luciferase assay, MVLN ce lls were withdrawn from estrogen six days prior to passage and then plated in medium and fed treatment media containing the test chemical in ethanol (tested concentrations were not reported, solvent concen tration <1%). Relative luciferase activity (%) was calculated against 17 β -estradiol (1 nM) as the standar d, and relative estrogenic potency was
determined as the ratio between the minimal concentration of 17 β -estradiol needed for a maximal response and the minimal dose of the te st chemical needed to achieve a maximal response. The relative estrogenic activity of a test chemical in solution was estimated as the product of the relative estrogenic potency and th e relative luciferase ac tivity. In the MVLN assay, o-PP had a relative estrogenic potency of 0.0054 compared to 1 nM 17 β -estradiol, and a relative luciferase activity of 23%. The relative es trogenic activity of o-PP was calculated to be

0.00012.

Subchronic Oral Toxicity in Non-Rodents (Dog; OCSPP 870.3150)

In a subchronic toxicity study in dogs ( MRID 41656401 ), o-PP (99.77% purity) dissolved in peanut oil was administered by gastric intubation to Beagle dogs (2/sex/dose group) at doses of 0, 100, 200, or 400 mg/kg/day for four weeks; the 400 mg/kg/day dose was reduced to 300 mg/kg/day on Day 5 due to excessive emesis. Organ weights were determined for the kidneys and liver; histopathological examinations were co nducted on these tissues as well as the ovaries, testes, pituitary, thyroid, adre nals, urinary bladder, epididym is, prostate, mammary gland, and
cervix.

Dose-related emesis was noted in all treated animals with the high-dose group exhibiting the most severity; occurrence of emesis by anim al-days for males/females was 0/0, 2/3, 12/11 andO-PHENYLPHENOL FINAL

Page 45 of 56 15/17 for the control, low- , mi d- and high-dose groups, respectivel y. Body weight gains in all study animals were variable, but treated animals ge nerally gained less (or lost) weight during the study period. No treatment-related differences were noted in food consumption. No treatmentrelated differences were noted in the ophthalm ological examination, urinalysis, hematology, or clinical chemistry results. Finally, there were no treatment-related changes in the absolute or relative weights of the liver a nd kidneys, with no differences noted in gross or microscopic pathology.

The LOAEL is 200 mg/kg/day based on increased em esis and reduced body weight gain, and the NOAEL is 100 mg/kg/day.

Prenatal Development in Rodents (Rat; OSCPP 870.3700)

In a developmental toxicity study in rats ( MRID 00067616/00164362; MRID 92154037 ), o-PP (99.69% purity) was administered by gavage to mated SD rats (37/cont rol group; 27/low- and medium-dose groups; and 26/high-dose group) at doses of 0 (cottonseed oil), 100, 300, or 700 mg/kg/day on GDs 6-15, inclusive. The rats were sacrificed at GD 21 and the number and position of live, dead, and resorbed fetuses, and staining of apparent non -pregnant uteri, were determined along with liver weights. All fetu ses were weighed, measured, sexed, and examined for gross external alterations and cleft palate. One-third of the fetuses in each litter were
examined for visceral soft tissu e alterations by dissection; heads were examined after sectioning. The remaining two-thirds of the fetuses in each litter were examined fo r skeletal alterations.

There were no reported clinical observations related to treatment. Body weight gains during GDs 6-16 were decreased (NS) by 9 and 21% in the medium- and the high-dose groups, respectively, with overall body weight gain (GDs 6-21) decreases of 9 and 7%. Food consumption and food efficiency were slightly reduced in the medium- and high-dose groups during the dosing period. Decreased (NS) absolute and relative liv er weights were noted in the high-dose group ( ↓ 8 and 6%, respectively).

The maternal toxicity LOAEL is 300 mg/ kg/day and the NOAEL is 100 mg/kg/day based on decreased body weight gain, food consumption, a nd food efficiency. There were no treatmentrelated changes in the number of corpora lutea, implantation sites, early or late resorptions, number of live fetuses/litter, fetal sex ratio, or developmental abnormalities/malformations at any dose. The developmental toxicity LOAEL is >700 mg/kg/day and the developmental toxicity
NOAEL is ≥ 700 mg/kg/day.

Prenatal Development in No n-Rodents (Rabbit; OCSPP 870.3700)

In a developmental toxicity study in rabbits ( MRID 41925003 ), o-PP (99.88% purity) was
administered to female New Zealand White rabb its (18/control group; 16/low- and medium-dose groups; and 24/high-dose group) by ga vage at doses of 0 (corn oil), 25, 100 or 250 mg/kg/dayO-PHENYLPHENOL FINAL

Page 46 of 56 from GDs 7-19, inclusive. On GD 28, all survivin g does were sacrificed, a nd subject to detailed necropsy. In addition, the number and position of fetuses, the number of live and dead fetuses, the number and position of resorption sites, and th e number of corpora lutea in pregnant animals were recorded. All fetuses were sexed, we ighed, and examined for gross external
malformations. Each fetus was examined viscer ally by fresh dissection and the brain examined
via coronal section. All carca sses were eviscerated, cleare d, and stained for skeletal
examination.

Ten deaths occurred during the st udy; seven animals died spontaneous ly or were sacrificed in a moribund condition, and three animals were sacrificed due to evidence of a bortion. At least four of these deaths, in the high-dose group, were cons idered to be treatment-related and the abortion at the high dose also may have been related to treatment. A dose-related increase in the incidence of blood in the feces, urine, and/or cage pan was noted in the medium- and high-dose groups (3/medium and 6/high vs. l/control), and increased incidences of decreased feces and perineal soiling may also of have been related to treatment. Ther e were no effects of treatment on body weight or body weight gain , or on absolute or relativ e kidney or liver weight.
Treatment-related alterations in microscopic kidney structure ( e.g. , inflammation and tubular degeneration) were noted only in the high-dose an imals. There appeared to be no treatmentrelated effects on the number of corpora lutea, impla ntation sites, live or dead fetuses, or early or late resorptions. Mean fetal weight values, fe tal viability, and fetal sex ratio were similar between control and treated groups. There were no statistical or biol ogically-significant,
treatment-related differences in the incidence of any fetal malformation or variation in any of the dose groups.

The maternal LOAEL is 100 mg/kg/day, based on treatment-related microscopic kidney alterations, and the maternal NOAEL is 25 mg/kg/day. The LOAEL for developmental toxicity is not determined, and the NOAEL is ≥ 250 mg/kg/day.

Two-Generation Reproduction in Rodents (Rat; OCSPP 870.3800)

In a two-generation reproduction study in rats ( MRIDs 40976501 and 41729401 ), o-PP (99.86% and 99.47% purity; Weeks 0-38 and 38-study terminati on, respectively) was administered to CD SD rats (35/sex/dose group) in the diet at nomin al doses of 0 (acetone and corn oil), 40, 140, and 490 mg/kg/day (actual intakes 36, 125, and 457 mg/kg/ day). The F0 parental animals were given the test diets 15 weeks prior to mating to pr oduce the F1 offspring and continued throughout mating, gestation, and lactation, with further expo sure of the offspring after weaning. The F1 parental animals were not mated until 10 weeks af ter they were selected from the F1b litters (selected at weaning, 21 days of age). Trea tment continued throughout mating, gestation, and lactation, with further exposure of the offspring after weaning. The estrous cycle was examined from vaginal smears taken for three weeks from F0 and F1 (10/dose) prior to mating. The estrous cycle was classified as leukocyte (diestru s), proestrus, proestrus/cornified, and cornifiedO-PHENYLPHENOL FINAL

Page 47 of 56 (estrus). The organs weighed at sacrifice includ ed the liver, kidneys, testes, and ovaries. Gross and microscopic pathological examinations we re conducted on these organs as well as the pituitary, urinary bladder, epididymis, prostate , seminal vesicles, vagina, uterus, and cervix.

There were no treatment-related c linical signs of toxicity in an y of the treated groups, including adults and pups. There was no treatment-related in crease in mortality; however, one animal in the medium-dose group probably died as a result of uremia or kidney failure that may be treatment-related. Body weights were decrease d (p<0.05) during the pre-mating periods in highdose F0 males and females ( ↓ 7-8% and 6-7%, respectively) a nd the high-dose F1 males and females ( ↓ 11-12% and 7-12%, respectively). Decreased body weights were also reported for the high-dose groups (both F0 and F1) during th e rest period between the F1a and F1b ( ↓ 8-12% and 6-7% in males and females, respectively) and the F2a and F2b ( ↓ 12-13% and 9-10% in males and females, respectively) breedings. Although body weights were occasionally decreased (p<0.05) in females during the gestation and lact ation periods, these decreases were generally <10% and body weight gains were not affect ed. Food consumption also was decreased (significance not reported) in the high-dose F0 and F1 groups during the F1a - F1b and F2a - F2b rest periods.

No treatment-related effects were observed in the reproductive performances (male or female mating indices, male or female fertility indices , gestation index, gestati on length, live birth index, viability index, lactation index, parturition, or fe tal sex ratio). There was no treatment-related effect on estrus cycling measured in the F0 and F1 rats during th e 14-day period prior to breeding, and no treatment-re lated changes in absolute or relative weights of the testes or ovaries
were seen in the F0 and the F1 adults. Relativ e kidney weights were increased (p<0.05) in highdose F0 and F1 males, with no change in absolute weights. Other changes in kidney, liver, and gonad weights were not considered to be related to treatment (only kidney data was provided). No treatment-related histopathological lesions were seen in the testes, epididymides, seminal vesicle, prostate, ovaries, uter us, vagina, cervix, and pituitary gl and of the F0 and F1 adults.
Increased incidences of renal calculi (p<0.05) in high-dose F0 males and in urinary bladder calculi (NS) in the medium- and high-dose F0 ma les and the high-dose F1 males were observed. In addition, there was an increased (p<0.05) inci dence of transitional cell hyperplasia in the urinary bladder in high-dose F0 males and females and in high-dose F1 males.

At the high-dose, body weights were decrea se (p<0.05) by 13 and 18% on PNDs 14 and 21, respectively, for the F1b pups, by 6 and 12% on PNDs 14 and 21 for the F2a pups, and by 12% on PND 21 for the F2b pups.

The parental NOAEL is 40 mg/kg/day (36 mg/kg/ day actual), and the parental LOAEL is 140 mg/kg/day [125 mg/kg/day actu al; based on decreases in body weight, increases in relative kidney weights, in urinary bladder and kidney tran sitional cell hyperplasia, in renal hemorrhage, and in pyelonephritis starting at the high dose, and on an increase in urinary bladder calculiO-PHENYLPHENOL FINAL

Page 48 of 56 starting at the medium dose (gross obse rvations only)]. The reproductive NOAEL is 490 mg/kg/day (457 mg/kg/day actual; HDT), and the LOAEL is not determined. The developmental NOAEL is 140 mg/kg/day, and th e developmental LOAEL is 490 mg/kg/day based on decreased body weight in pups.

In a subsequent two-generation reproduction study in rats ( MRID 43928801 ), o-PP ( ≥ 99.5% purity) was administered continuou sly in the diet to SD rats (30/sex/dose group) at nominal doses of 0, 20, 100, or 500 mg/kg/day for two c onsecutive generations. Test material concentrations were adjusted weekly duri ng the pre-mating period based on group mean body weight and food consumption for each dose group. During the gestation and lactation periods, the females from each dose group received the same dietary concentrations that were supplied during the last week of pre- mating. The actual doses duri ng the lactation phase were approximately twice the doses received during the pre-mating and gestation phases because the dose was not adjusted and food consumption increas ed. The P generation animals were exposed to the test diets, beginning at approximately eight weeks of age, for ten weeks prior to mating to produce the F1a litter. After a two-week rest period, the P animals were mated again to produce the F1b litter. The F1b offspring (30/sex/dose group) selected to be parents of the next generation were fed the same test diet concentra tions as their parents fo r twelve weeks prior to mating to produce the F2 litters. The organs wei ghed at sacrifice included the kidneys, testes,
and ovaries. Gross and microsc opic pathological examinations were conducted on these organs as well as the pituitary, urinary bladder, ep ididymis, coagulating gland, prostate, seminal vesicles, vagina, uterus, and cervix.

One P generation high-dose male was found dead on Day 173 due to kidney failure; this death
was attributed to treatment. Five other rats found dead and six euthanized in extremis were
attributed to other non-treatment causes. Additionally at this dos e, an increase in the incidence of urine staining was noted in the P and F1 genera tion males. This finding was associated with the gross and microscopic lesions of the urinary tract in these animals.

In the high-dose P generation females, pre- mating body weights were decreased (p<0.01; ↓ 5-7%) on Days 21, 42, and 70. Additionally, overall bod y weight gains (Days 0-175, male or 0-70, female) were decreased (NS) in th e P generation males and females ( ↓ 12 and 19%, respectively). In the F1 generation, body weights were decr eased (p<0.01) throughout the entire pre-mating period in males and females ( ↓ 9-13%, males and ↓ 7-12%, females, respectively). Overall body weight gains also were decreased (NS) in the F1 generation males and females ( ↓ 10 and 8%, respectively). No differences in overall (GD 0-20) body weight gains were noted in either female generation during gestati on. Body weights were decreased (p<0.01 or 0.05) in the highdose P and F1 generation females throughout the gestation periods for the F1a, F1b, F2a, and F2b litters ( ↓ 5-9%, except for GD6 during gestation of the F2b litter; ↓ 11%). These decreases were considered a continuation of the lower body weights observed during pre-mating in the P and F1 generation females. Body weights were decreased (p<0.01 or 0.05) in the P generationO-PHENYLPHENOL FINAL

Page 49 of 56 high-dose females throughout lactation for both the F1a and F1b litters ( ↓ 6-9%). Additionally in the high-dose F1 generation females, body weight s were decreased (p<0.01) during LD 0-14 of the F2a litter and decreased (p<0.05) during LD 0-7 of the F2b litter ( ↓ 8%). At this dose, absolute food consumption was increased during lactation for both litters in both generations.

Treatment-related microscopic lesions noted in the urinary tract of the high-dose P generation males included: (i) simple hyperplasia, nodular/papillary hyperplasia, calculi, and chronic inflammation in the urinary bladder; (ii) chro nic active inflammation a nd debris in the renal pelvis in the kidney; and (iii) dilation and hype rplasia in the ureter. Similar findings were observed in the high-dose F1 generation males. No organ weight changes due to treatment were observed. Gross necropsy findings were limited to an increased incidence of urinary bladder calculi in the high-dose P and F1 generation male s. No treatment-related histopathological
lesions were seen in the test es, epididymides, seminal vesicl e, prostate, coagulating gland, ovaries, uterus, cervix, vagina, or pituitary glands of the P and F1 adults. In addition, no adverse
effects of treatment were noted at the low or medium dose in either generation.

The LOAEL for parental toxicity is 500 mg /kg/day based on decrea sed body weights, body weight gains, and effects on the urinary tract (bladder, kidneys and ureter) in males. The NOAEL for parental toxic ity is 100 mg/kg/day.

There were no compound-related ef fects on estrous cycle duration or the number of days until insemination in either generation. No treat ment-related effects on the mating, fertility, or gestation indices were observed in either ge neration. There were no effects of treatment on gestation duration in either gene ration. The LOAEL for reproduc tive toxicity is not observed, and the NOAEL for reproductive to xicity is 500 mg/kg/day.

No clinical signs of toxicity we re observed in the pups of either generation from birth to PND 21.
No treatment-related effects on the number of litter s, number stillborn, litter size, sex ratio, or live birth, viability, or lactation indices were observed in either generation. There were no treatment-related gross lesions noted in the pups. At the high dose, overall (PND 1-21) pup body weight gains were decreased (NS) in both generations compared to control ( ↓ 12-14%). Body weights at this dose were decreased (p<0.01 or 0.05) in the F1 generation on PND 21 ( ↓ 10-12%) and in the F2 generation on PNDs 14 ( ↓ 6-7%) and 21 ( ↓ 11-12%). No treatment-related effects
were noted at the low or medium dose in the pups. The LOAEL for offspring toxicity is 500 mg/kg/day based on decreased pup body weight s and body weight gains. The NOAEL for offspring toxicity is 100 mg/kg/day.

Chronic Oral Toxicity/Carcinogenici ty in Rodents (Rat; OCSPP 870.4300)

In a chronic toxicity/carci nogenicity study in rats ( MRIDs 43954301, 44832201, 44852701 ), oPP ( ≥ 99.5% purity) was administered in the diet to CDF [F-344]/BR rats (50/sex/dose group) at nominal concentrations of 0, 800, 4000 and 8000 ppm in males and 0, 800, 4000, andO-PHENYLPHENOL FINAL

Page 50 of 56 10,000 ppm in females (equivalent to 39, 200, a nd 402 mg/kg/day for males and 49, 248, and 647 mg/kg/day for females) for two years. An in terim-sacrifice group of twenty animals/sex for control and high-dose groups and ten animals/se x for the low- and medium-dose groups were
sacrificed at twelve months. At study termination, surviving rats in all groups were sacrificed for gross and histopathological evaluation. The orga ns weighed at sacrifi ce included the adrenals, liver, kidneys, testes, ovaries, a nd thyroid. Gross and microscopi c pathological examinations were conducted on these organs as well as the pi tuitary, urinary bladder, epididymis, prostate, seminal vesicles, mammary gl and, uterus, and vagina.

There was no treatment-related effect on mortalit y observed in this study. There was an increase in abnormal color urination and red staining in high-dose males, brown staining in medium- and high-dose females, and urine staining in high-dose males and all treated females. Body weights in the medium-dose animals were decreased (p <0.05) in males and females in the two-year oncogenicity group (no decrease in the one-year interim sacrifice group) at Weeks 13, 52, and 78 ( ↓ 3-5%), with weights comparable to control at Week 104. Body weight gains in the mediumdose animals were generally >90% of control. Body weights in the high-dose animals were decreased (p<0.05) in males and females in the one-year interim sacrifice group ( ↓ 7-9%) and two-year oncogenicity group at Weeks 13, 52, 78, and 104 ( ↓ 9-15%). Body weight gains in the high-dose animals were decreased by 11-26%, comp ared to the control. Food consumption was generally decreased in th e two-year carcinogenicity group at the high-dose ( ↓ 4-11%), and was decreased sporadically in the medium-dose animals ( ↓≤ 8%).

Organ weight effects at study termination in high-dose male s included decreased (p<0.05) absolute and relative liver weight ( ↓ 19 and 10%, respectively) a nd absolute kidney weight ( ↓ 13%), and increased (p<0.05) absolute and relative testes weights ( ↑ 34 and 46%, respectively). No related pathology was observed to account fo r organ weight differences, except for the kidneys. No treatment-related changes were seen in absolute or relative weights of the ovaries, thyroid, or the adrenal glands.

Gross pathological observations at study terminat ion included an increase in observed masses in the urinary bladder of high-dose males and an in creased incidence of ki dneys with pitted zones in high-dose females. Non-neoplastic effects obs erved included increased incidences of renal calculi in high-dose males at the 12-month s acrifice (kidneys) a nd the 24-month study termination (urinary bladder). Increased hyperplasia of the urin ary bladder was present at the 12- and 24-month sacrifices in high-dose males (and high-dose females at study termination), along with an increase in congestion, hemorrhage, mineralization, and necrosis of the urinary bladder at study termination in high-dose males. High-dose females had an increase in hyperplasia of the kidney, as well as increased in farct, acute inflammation and mineralization of the kidney. High-dose males and females also had an increase in cysts of the kidney at study termination. Relevant neoplasti c observations were confined to the urinary bladder with an increased incidence of papilloma and transitional cell carcinomas in high-dose males noted at theO-PHENYLPHENOL FINAL

Page 51 of 56 12-month sacrifice and the 24-month study termination in both the medium- and high-dose males (transitional cell carcinoma). Th ese findings were not present in females. No treatment-related effects on histopathology were noted in the test es, epididymides, seminal vesicle, prostate, ovaries, uterus, vagina, mammary glands, thyroid, pituitary, or adrenal glands at any dose.

The systemic toxicity LOAEL is 4000 ppm ( 200 mg/kg/day for males and 248 mg/kg/day for females) based on decreased body weight and bod y weight gain, decreased food consumption and food efficiency, and increased cl inical and gross pathological si gns of toxicity. The systemic toxicity NOAEL is 800 ppm ( 39 mg/kg/day for males and 49 mg/kg/day for females).

Carcinogenicity in Rodents (Mouse; OCSPP 870.4200)

In a carcinogenicity study in mice ( MRID 43545501 ), o-PP (99.88% purity) was administered to B6C3F1 albino mice (60/sex/dose group) in the diet at concentrations equi valent to doses of 0, 250, 500, and 1000 mg/kg/day for 24 months. Interim sacrifices were con ducted after twelve months of dosing with 10 mice/sex/dose group. At study termination, all surviving mice in all groups were sacrificed for gross and histopat hological evaluation. Th e organs weighed at sacrifice included the adrenals, liver, kidneys, and testes. Gro ss and microscopic pathological examinations were conducted on these organs as well as the pituitary, t hyroid, urinary bladder, epididymis, prostate, seminal vesicles, mammary gland, uterus, and vagina.

There was no treatment-related effect on morta lity in the interim-sacrifice or oncogenicity groups, and no clinical signs of toxicity. Decreased (p<0.05) body weights were observed in the high-dose males by Day 147 with de creases ranging from 3-9% between Days 147-287. Further body weight decreases (p<0.05) reached 10% in the high-dose males at Day 315, and ranged from 10-18% from Day 315-728 (study terminati on). Decreased (p<0.05) body weights were observed in the medium-dose males by Day 231 with decreases ranging from 4-8% between Days 231-728. Overall body weight gains were d ecreased by 16 and 27% in the medium- and high-dose males; no effects on body we ight or body weight gain occu rred in the low-dose males. Intermittent decreases (p<0.05) in body weights were noted in all dose groups in female mice through Day 91. Consistent body weights decreases (p<0.05) were observed in the high-dose females by Day 147 ranging from 6-8% between Days 147-231. Further body weight decreases (p<0.05) reached 11% in the high-dose female s at Day 259, and ranged from 11-25% from Day 259-728. Consistent body weights decreases (p <0.05) were observed in the medium-dose females by Day 343 with decreases ranging fr om 7-9% between Days 343-427; further body weight decreases (p<0.05) ra nged from 10-18% from Day 455- 728. Generally non-significant body weight decreases were presen t in the low-dose females ( ≤ 6%) between Days 119-623; greater decreases (p<0.05; ↓ 9-11%) were observed at Days 651-707, with a decrease in body weight (NS, ↓ 6%) at Day 728. Overall body weight gain s were decreased by 10, 25, and 38% in the low-, medium- and high-dose females during the study. No consiste nt, or discernible,
treatment-related effects on food c onsumption or food efficiency we re observed during the study.O-PHENYLPHENOL FINAL

Page 52 of 56 No consistent, treatment-related findings were ob served in the hematology, clinical chemistry, or urinalysis findings. There were increases (p<0.05 or NS) in absolute and relative liver weights at the interim (12-month) and terminal (24-month) sacrifices in all treated males and females; increases were non-dose-dependent, except for interim-sacrifice females, and the increases were greater after the 12-mont h treatment compared to the 24-month treatment. Absolute adrenal weights were increased in the treated males, a nd decreased in treated females, in a non-dosedependent manner at the terminal sacrifice. Sple en weights (absolute and relative) in males and females were reduced in all treated groups (ex cept low-dose males); increases in females were non-dose-dependent. No treatment-related changes we re seen in absolute or relative weights of the testes at any dose.

Non-neoplastic observations showed an accentuated lobular pattern of the liver of all treated
animals. There was an increased incidence in hepatic tumors in high-dos e males at the interim sacrifice, and in medium- and high-dose males at the terminal sacrif ice in the form of hepatocellular adenomas; similar tumors with a lesser incidence were obs erved in the mediumand high-dose females at the terminal sacrifi ce. No treatment-relate d effects on histopathology were noted in the testes, epididymides, semina l vesicle, prostate, ova ries, uterus, mammary glands, thyroid, pituitary, or adrenal glands at any dose.

A NOAEL was not observed. The systemic toxicity LOAEL is ≤ 250 mg/kg/day, based on increased liver weights, and decr eased spleen weights, and gross observations in the livers of all
treated animals.

Chronic Oral Toxicity in No n-Rodents (Dog; OCSPP 870.4100)

In a chronic toxicity study in dogs ( MRID 41656401; 1990 ), o-PP (99.77% purity) was administered via gastric intubation to Beagle dogs (2/sex/dose group) at doses of 0 (peanut oil), 30, 100, or 300 mg/kg/day for one year. Organ we ights determined included adrenals, kidneys, liver, ovaries, testes, pituitary, and thyroid; hi stopathological examinations were conducted on
these tissues as well as the ur inary bladder, epididymis, prostate, mammary gland, and cervix.

Two dogs in the high-dose group died on study due to gavage error. Dose-related emesis was noted in all treated animals, with the high-dose group exhibiting the most severity (emesis was generally similar to control at the two lower doses ). Body weight gains in the high-dose females were decreased (NS; ↓ 7-16%) at all time-points through stud y termination. Body weight gain
decreases in low- and medium-dose females at Days 92 and 176 were not observed at study termination. Food consumption was slightly in creased (NS; 8-13%) in the high-dose group, with a lower food efficiency in the high-dose females. No treatment-related differences were noted in the ophthalmological examination, urinalysis, hematology, or clinical chemistry results. There were no treatment-related changes in the absolute or relative weights of the adrenals, kidneys, liver, ovaries, testes, pituitary, and thyroid. Ther e were also no treatment -related effects on gross or microscopic pathology, including in the te stes, epididymides, prostate, ovaries, uterus,O-PHENYLPHENOL FINAL

Page 53 of 56 mammary glands, thyroid, pituitary, or adrenal glands at any dose, and no evidence for oncogenicity due to o-PP treatment.

The LOAEL is 300 mg/kg/day based on increa sed emesis, reduced body weight gain, and reduced food efficiency, and the NOAEL is 100 mg/kg/day.

Developmental Toxicity (Rat) and Dominant Lethal (Mouse) Studies

In a published non-Guideline study, Kaneda et al . (1978) dosed pregnant Wistar rats (14-weeks of age) with o-PP (99.7%) at 0, 150, 300, 600, and 1200 mg/kg/day in distilled water with 5% gum arabic by gavage on GDs 6 through 15; dams were sacrificed on GD 20. The number of dams assigned to each dose group was not stated directly; apparently, the control, low-, and medium-low dose groups had twenty animals each, with eighteen animals in the medium-high dose group and eleven in the high-dose group. The uterine contents were examined for implantation sites and live or dead fetuses; li ve young were inspected externally and weighed individually. The pups were divided into two gr oups; half of the pups were fixed in 95% alcohol and prepared for staining with alizarin red S for skeletal examination and the remaining pups
were fixed in Bouin's fluid for internal examin ation according to a m odified method of Wilson.

Ataxia was observed after dosing in dams administered o-PP at ≥ 300 mg/kg/day; the duration of ataxia was reported as lasting se veral hours with severity increas ing in a dose-related manner. Ten of eleven dams died after 3-9 days of treatment in the high-dose group; no clinical signs of toxicity were noted at the low dose (150 mg /kg/day). Body weight gains on GDs 9, 12, 15, and 20 were decreased in the medium-low (p<0.05, 0.01, or 0.001; ↓ 17-28%) and medium-high (p<0.001; ↓ 46-62%) dose groups. Body weight gain also was decreased in the single surviving animal in the high-dose group (not statistically assessed). No treatment-related changes were seen in the number of implantation sites, resorpti on rates, or number of live fetuses/litter in the low and medium-low dose groups. Fetal reso rptions were increased (p<0.01; 25.7% vs. 13.9% control) at the medium-high dose, and pup wei ghts were decreased (p<0.01) by 6-8%. Fetal resorptions were further increa sed (not statistically assessed; 38.5%) in the one remaining highdose dam, with further decreases in pup weights. Soft tissue and skeletal examinations did not reveal any treatment-related effects.

The same authors dosed male C3 H mice (10-weeks of age; 15/dose group) with o-PP at 0, 100 or 500 mg/kg/day in distilled water with 5% gum arab ic for 5 consecutive days. Immediately after the administration, each male was mated with two unt reated virgin females. After one week, the females were removed and replaced with two other untreated virgin females (this mating procedure was repeated for six weeks). Female s were sacrificed on GD 12 or 13, and scored for the numbers of corpora lutea, implants, living em bryos, and early or late embryonic deaths. The frequency of dominant-lethal mutations was calcu lated. No increase was seen in the frequency of induced dominant-lethal mutations at any dose or mating period. The mean numbers ofO-PHENYLPHENOL FINAL

Page 54 of 56 corpora lutea, implants, live embryos per pregnanc y, and incidences of early embryonic deaths in the treated groups were not different from control.

Freshwater Fish Reproduction (Fathead minnow)

The 21-day short-term reproduction assay of 2- phenylphenol (o-PP) with fathead minnows ( Pimephales promelas) was conducted under flow-through conditions ( MRID 48135404 ). Adult fish, 6 spawning groups (1 male and 1 female in each group; ca . 6 months old), were exposed to 2-phenylphenol (99.9% purity) at nominal con centrations of 0 (c ontrol), 1.0, 5.0, 50.0 and 500 μg a.i./L; average (MEAN)-measured concentr ations were <LOQ (not reported), <LOQ, 4.03, 36, and 293 μg a.i./L. A positive control (17 α ethynylestradiol; 10 ng/L) was also included. The test system was maintained at 24.7 to 25.5 o C and a pH of 7.50 to 7.92.

The study consisted of a 21-day pre-exposure ph ase followed by a 21-day exposure phase. The same 6 spawning groups were used for each phase. In the pre-exposure phase, the study measured fecundity (as represented by the numbe r of spawnings, number of eggs, and number of eggs per brood) and fertility (as represented by egg hatch) to establish fish with a record of reproductive success. Fish that did not have sufficient egg production or egg hatch during the pre-exposure phase were excluded from the expos ure phase. This resulted in variable n sizes across the control and treatment groups (n size is 3, 4, 4, 4, 3, and 4 for the negative control, positive control, <LOQ, 4.03, 36, and 293 μg/L treatme nt groups, respectively). In the exposure phase, the study measured fecundity (same as above), fertility (same as above), plasma vitellogenin (VTG) and gonadosomatic index (GSI).

Mortality was observed in the study with the highest number of mortalities occurring in the negative control. All mortalities occurred during the pre-exposure phase. No significant effects were observed in fertility, plasma VTG or GSI fo r either the male or female fish. Mean number of eggs per female was 274 in the control, and 220, 268, 166, 94 and 15.5 in the positive control, low, mid-low, mid-high and highest treatmen t group, respectively. There was a significant
decrease of 65.7% and 94.3% in measures of fec undity (as represented by the number of eggs per female) in the 36 μg/L (p = 0.0081) and 293 μg/L (p = 0.0013) treatment groups compared to the control. It is noted that there were only 3 breeding pair replicates available in the negative control during the exposure phase and variability in fecundity output was observed (total number of eggs per female in control was 328, 0 and 495 eggs/female). However, in the highest treatment (n=4 replicates), total number of e ggs ranged from 0-22, and was 131, 151 and 0 in the three replicates in the mid-high treatment replicates (n=3).O-PHENYLPHENOL FINAL

Page 55 of 56 APPENDIX 3: References Not Utilized in the o -Phenylphenol WoE Analysis

In 2009, after public review and comment, a fina l list of 67 chemicals and schedule for issuing
Test Orders for the EDSP Tier 1 screening battery was made av ailable in a Federal Register Notice issued October 21, 2009 (74 FR 54422). The ag ency’s review of the initial data submitted as “other scientifically releva nt information (OSRI) was provided in the Report of the Endocrine Disruptor Review Team (USEPA, 2010).

Beginning in 2011, the agency has reviewed data cited as “OSRI which included Part 158 studies previously submitted to the agency for registra tion/reregistration, published literature articles and/or Tier 1 assays. The agency also c onducted a more recent search (2009 to 2014) of
available scientific literature fo r any additional relevant informa tion for their weight of evidence (WoE) evaluations. These articles were evalua ted in accordance with the agencies Evaluation Guidelines for Ecological Toxicity Data in Open Literature, May 2011 ( http://www.epa.gov/pesticides/s cience/efed/policy_guidance/team _authors/endangered_species _reregistration_workgroup/PDF_ro t/esa_evaluation_open_literature.pdf ) and the 2012 Guidance for considering and Using Open Literature To xicity Studies to Suppor t Human Health Risk Assessment ( http://www.epa.gov/pesticides /science/lit-studies.pdf).

The following published and unpublished references were considered for use in the WoE analysis for O-Phenylphenol but were not utilized due to one or more of the following reasons: 1) the article was not available in English; 2) the compound of interest wa s not used in the study; 3) the test material was not adequately describe d; 4) a formulated end- use product or mixture of chemicals was utilized as the test material; 5) only acute mortality toxicity data were provided; 6) the experimental conditions were not adequate ly described; 7) only an abstract of the study was available; 8) the reference is a review article or book chapte r and does not contain primary study data; 9) insufficient information was availabl e to adequately assess th e validity of the study results; 10) the 40 CFR Part 158 guideline study wa s classified as unacceptable/inadequate; 11) the study dealt only with non- EDSP assay development; 12) no specific endocrine-related endpoints were assessed in the study; and 13) th e study contained only da ta on invertebrates.

Caspers, N. (2001) Life cycle test with water fleas-Daphnia Ma gna. Testing laboratory: Bayer AG, WG-UWS, Institute of Environmental Analysis Evaluation, D-51368 Leverkusen. Study number, 51 A/88. Study Sponsor: Bayer AG. (MRID 48135402)

Caspers, N. (2001) Preventol 0 extra daphnia magna reproduction test. Tes ting laboratory: Bayer AG, WG-UWS, Institute of Environmental Analysis Evaluation, D-51368 Leverkusen. Study number, 1092 A/01 DL. Study Spons or: Bayer AG. (MRID 48135403)

Itoh, S., Ueda, H., Naasaka, T., Nakanishi, G., and Sumitomo, H. (2000) Evaluating variation of estrogenic effect by drinking water ch lorination with the MVLN assay. Water Science and Technology 42 (Nos. 7-8): 61-69.O-PHENYLPHENOL FINAL

Page 56 of 56 NTP Technical Report: Toxicology and carcinogenesis studies of orthophenylphenol (CAS No. 90-43-7) alone and with 7,12-Dimethylbenz(a)a nthracene (CAS No. 57-97-6) in Swiss CD-1 mice (dermal studies). NTP TR 301 (1986).

Soto, A., Fernandez, M., Luizzi, M., Oles Kara sko, A., and Sonnenschein, C. (1997) Developing a marker of exposure to xenoestro gen mixtures in human serum. Environmental Health Perspectives 105(3): 647-654.